Anti-PRO9821 antibodies

ABSTRACT

Compositions and methods are disclosed for stimulating or inhibiting angiogenesis and/or cardiovascularization in mammals, including humans. Pharmaceutical compositions are based on polypeptides or antagonists thereto that have been identified for one or more of these uses. Disorders that can be diagnosed, prevented, or treated by the compositions herein include trauma such as wounds, various cancers, and disorders of the vessels including atherosclerosis and cardiac hypertrophy. In addition, the present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

RELATED APPLICATIONS

This is a continuation application claiming priority under 35 USC § 120to U.S. application Ser. No. 10/081,056, filed Feb. 20, 2002, nowabandoned which is a continuation of International application NumberPCT/US01/21735, filed Jul. 9, 2001, which is a continuation-in-part ofInternational application Number PCT/US01/19692, filed Jun. 20, 2001,which claims priority under 35 USC § 119 to U.S. provisional applicationSer. No. 60/232,887, filed Sep. 15, 2000, the entire disclosures ofwhich are hereby incorporated by reference.

1. FIELD OF THE INVENTION

The present invention relates to compositions and methods useful for themodulation (e.g., promotion or inhibition) of angiogenesis and/orcardiovascularization in mammals in need of such biological effect. Thepresent invention further relates to the diagnosis and treatment ofdisorders involving angiogenesis (e.g., cardiovascular as well asoncological disorders).

2. BACKGROUND OF THE INVENTION

2.1. Angiogenesis

Angiogenesis, defined as the growth or sprouting of new blood vesselsfrom existing vessels, is a complex process that primarily occurs duringembryonic development. Under normal physiological conditions in adults,angiogenesis takes place only in very restricted situations such as hairgrowth and wounding healing (Auerbach, W. and Auerbach, R., 1994,Pharmacol Ther 63(3):265–3 11; Ribatti et al., 1991, Haematologica76(4):3 11–20; Risau, 1997, Nature 386(6626):67 1–4). Unregulatedangiogenesis has gradually been recognized to be responsible for a widerange of disorders, including, but not limited to cardiovasculardisease, cancer, rheumatoid arthritis, psoriasis and diabeticretinopathy (Folkman, 1995, Nat Med 1(1):27–31; Isner, 1999, Circulation99(13): 1653–5; Koch, 1998, Arthritis Rheum 41(6):951–62; Walsh, 1999,Rheumatology (Oxford) 38(2):103–12; Ware and Simons, 1997, Nat Med 3(2):158–64).

2.2. Cardiac Disorders and Factors

Heart failure affects approximately five million Americans, and newcases of heart failure number about 400,000 each year. It is the singlemost frequent cause of hospitalization for people age 65 and older inthe United States. Recent advances in the management of acute cardiacdiseases, including acute myocardial infarction, are resulting in anexpanding patient population that will eventually develop chronic heartfailure. From 1979 to 1995, hospitalizations for congestive heartfailure (CHF) rose from 377,000 to 872,000 (a 130 percent increase) andCHF deaths increased 116 percent.

CHF is a syndrome characterized by left ventricular dysfunction, reducedexercise tolerance, impaired quality of life, and markedly shortenedlife expectancy. The sine qua non of heart failure is an inability ofthe heart to pump blood at a rate sufficient to meet the metabolic needsof the body's tissues (in other words, there is insufficient cardiacoutput).

At least four major compensatory mechanisms are activated in the settingof heart failure to boost cardiac output, including peripheralvasoconstriction, increased heart rate, increased cardiac contractility,and increased plasma volume. These effects are mediated primarily by thesympathetic nervous system and the renin-angiotensin system. See,Eichhorn, American Journal of Medicine, 104: 163–169 (1998). Increasedoutput from the sympathetic nervous system increases vascular tone,heart rate, and contractility. Angiotensin II elevates blood pressureby 1) directly stimulating vascular smooth muscle contraction, 2)promoting plasma volume expansion by stimulating aldosterone andantidiuretic hormone secretion, 3) stimulating sympathetic-mediatedvascular tone, and 4) catalyzing the degradation of bradykinin, whichhas vasodilatory and natriuretic activity. See, review by Brown andVaughan, Circulation, 97: 1411–1420 (1998). As noted below, angiotensinII may also have directly deleterious effects on the heart by promotingmyocyte necrosis (impairing systolic function) and intracardiac fibrosis(impairing diastolic and in some cases systolic function). See, Weber,Circulation, 96: 4065–4082 (1998).

A consistent feature of congestive heart failure (CHF) is cardiachypertrophy, an enlargement of the heart that is activated by bothmechanical and hormonal stimuli and enables the heart to adapt todemands for increased cardiac output. Morgan and Baker, Circulation, 83:13–25 (1991). This hypertrophic response is frequently associated with avariety of distinct pathological conditions such as hypertension, aorticstenosis, myocardial infarction, cardiomyopathy, valvular regurgitation,and intracardiac shunt, all of which result in chronic hemodynamicoverload.

Hypertrophy is generally defined as an increase in size of an organ orstructure independent of natural growth that does not involve tumorformation. Hypertrophy of the heart is due either to an increase in themass of the individual cells (myocytes), or to an increase in the numberof cells making up the tissue (hyperplasia), or both. While theenlargement of an embryonic heart is largely dependent on an increase inmyocyte number (which continues until shortly after birth), post-natalcardiac myocytes lose their proliferative capacity. Further growthoccurs through hypertrophy of the individual cells.

Adult myocyte hypertrophy is initially beneficial as a short termresponse to impaired cardiac function by permitting a decrease in theload on individual muscle fibers. With severe, long-standing overload,however, the hypertrophied cells begin to deteriorate and die. Katz,“Heart Failure”, in: Katz A. M. ed., Physiology of the Heart (New York:Raven Press, 1992) pp. 638–668. Cardiac hypertrophy is a significantrisk factor for both mortality and morbidity in the clinical course ofheart failure. Katz, Trends Cardiovasc. Med., 5:37–44(1995). For furtherdetails of the causes and pathology of cardiac hypertrophy see, e.g.,Heart Disease, A Textbook of Cardiovascular Medicine, Braunwald, E. ed.(W.B. Saunders Co., 1988), Chapter 14, “Pathophysiology of HeartFailure.”

On a cellular level, the heart is composed of myocytes and surroundingsupport cells, generically called non-myocytes. While non-myocytes areprimarily fibroblast/mesenchymal cells, they also include endothelialand smooth muscle cells. Indeed, although myocytes make up most of theadult myocardial mass, they represent only about 30% of the total cellnumbers present in heart. In response to hormonal, physiological,hemodynamic, and pathological stimuli, adult ventricular muscle cellscan adapt to increased workloads through the activation of ahypertrophic process. This response is characterized by an increase inmyocyte cell size and contractile protein content of individual cardiacmuscle cells, without concomitant cell division and activation ofembryonic genes, including the gene for atrial natriuretic peptide(ANP). Chien et al., FASEB J., 5: 3037–3046 (1991); Chien et al., Annu.Rev. Physiol., 55: 77–95(1993). An increment in myocardial mass as aresult of an increase in myocyte size that is associated with anaccumulation of interstitial collagen within the extracellular matrixand around intramyocardial coronary arteries has been described in leftventricular hypertrophy secondary to pressure overload in humans.Caspari et al., Cardiovasc. Res., 11: 554–558 (1977); Schwarz et al.,Am. J. Cardiol., 42: 895–903 (1978); Hess et al., Circulation, 63:360–371 (1981); Pearlman et al., Lab. Invest., 46: 158–164 (1982).

It has also been suggested that paracrine factors produced bynon-myocyte supporting cells may additionally be involved in thedevelopment of cardiac hypertrophy, and various non-myocyte derivedhypertrophic factors, such as, leukocyte inhibitory factor (LIF) andendothelin, have been identified. Metcalf, Growth Factors, 7: 169–173(1992); Kurzrock et al., Endocrine Reviews, 12: 208–217 (1991); Inoue etal., Proc. Natl. Acad. Sci. USA, 86: 2863–2867 (1989); Yanagisawa andMasaki, Trends Pharm. Sci., 10: 374–378 (1989); U.S. Pat. No. 5,573,762(issued Nov. 12, 1996). Further exemplary factors that have beenidentified as potential mediators of cardiac hypertrophy includecardiotrophin-1 (CT-1) (Pennica et al., Proc. Nat. Acad. Sci. USA, 92:1142–1146 (1995)), catecholamines, adrenocorticosteroids, angiotensin,and prostaglandins.

At present, the treatment of cardiac hypertrophy varies depending on theunderlying cardiac disease. Catecholamines, adrenocorticosteroids,angiotensin, prostaglandins, LIF, endothelin (including endothelin-1,-2, and -3 and big endothelin), and CT-1 are among the factorsidentified as potential mediators of hypertrophy. For example,beta-adrenergic receptor blocking drugs (beta-blockers, e.g.,propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol,penbutolol, acetobutolol, atenolol, metoprolol, carvedilol, etc.) andverapamil have been used extensively in the treatment of hypertrophiccardiomyopathy. The beneficial effects of beta-blockers on symptoms(e.g., chest pain) and exercise tolerance are largely due to a decreasein the heart rate with a consequent prolongation of diastole andincreased passive ventricular filling. Thompson et al., Br. Heart J.,44: 488–98 (1980); Harrison et al., Circulation, 29: 84–98 (1964).Verapamil has been described to improve ventricular filling and probablyreducing myocardial ischemia. Bonow et al., Circulation, 72: 853–64(1985).

Nifedipine and diltiazem have also been used occasionally in thetreatment of hypertrophic cardiomyopathy. Lorell et al., Circulation,65: 499–507 (1982); Betocchi et al., Am. J. Cardiol., 78: 451–457(1996). However, because of its potent vasodilating properties,nifedipine may be harmful, especially in patients with outflowobstruction. Disopyramide has been used to relieve symptoms by virtue ofits negative inotropic properties. Pollick, N. Engl. J. Med., 307:997–999 (1982). In many patients, however, the initial benefits decreasewith time. Wigle et al., Circulation, 92: 1680–1692 (1995).Antihypertensive drug therapy has been reported to have beneficialeffects on cardiac hypertrophy associated with elevated blood pressure.Examples of drugs used in antihypertensive therapy, alone or incombination, are calcium antagonists, e.g., nitrendipine; adrenergicreceptor blocking agents, e.g., those listed above; angiotensinconverting enzyme (ACE) inhibitors such as quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, and lisinopril; diuretics,e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, andindapamide; and calcium channel blockers, e.g., diltiazem, nifedipine,verapamil, and nicardipine.

For example, treatment of hypertension with diltiazem and captoprilshowed a decrease in left ventricular muscle mass, but the Dopplerindices of diastolic function did not normalize. Szlachcic et al., Am.J. Cardiol., 63: 198–201 (1989); Shahi et al., Lancet, 336: 458–461(1990). These findings were interpreted to indicate that excessiveamounts of interstitial collagen may remain after regression of leftventricular hypertrophy. Rossi et al., Am. Heart J., 124: 700–709(1992). Rossi et al., supra, investigated the effect of captopril on theprevention and regression of myocardial cell hypertrophy andinterstitial fibrosis in pressure overload cardiac hypertrophy, inexperimental rats.

Agents that increase cardiac contractility directly (iontropic agents)were initially thought to benefit patients with heart failure becausethey improved cardiac output in the short term. However, all positiveinotropic agents except digoxigenin have been found to result inincreased long-term mortality, in spite of short-term improvements incardiac performance. Massie, Curr. Op. in Cardiology, 12: 209–217(1997); Reddy et al., Curr. Opin. Cardiol., 12: 233–241 (1997).Beta-adrenergic receptor blockers have recently been advocated for usein heart failure. Evidence from clinical trials suggests thatimprovements in cardiac function can be achieved without increasedmortality, though documented improvements of patient survival have notyet been demonstrated. See also, U.S. Pat. Nos. 5,935,924, 5,624,806;5,661,122; and 5,610,134 and WO 95/28173 regarding the use ofcardiotropin-1 or antagonists thereof, or growth hormone and/orinsulin-like growth factor-I in the treatment of CHF. Another treatmentmodality is heart transplantation, but this is limited by theavailability of donor hearts.

Endothelin is a vasoconstricting peptide comprising 21 amino acids,isolated from swine arterial endothelial culture supernatant andstructurally determined. Yanagisawa et al., Nature, 332: 411–415 (1988).Endothelin was later found to exhibit various actions, and endothelinantibodies as endothelin antagonists have proven effective in thetreatment of myocardial infarction, renal failure, and other diseases.Since endothelin is present in live bodies and exhibits vasoconstrictingaction, it is expected to be an endogenous factor involved in theregulation of the circulatory system, and may be associated withhypertension, cardiovascular diseases such as myocardial infarction, andrenal diseases such as acute renal failure. Endothelin antagonists aredescribed, for example, in U.S. Pat. No. 5,773,414; JP Pat. Publ.3130299/1991, EP 457,195; EP 460,679; and EP 552,489. A new endothelin Breceptor for identifying endothelin receptor antagonists is described inU.S. Pat. No. 5,773,223.

Current therapy for heart failure is primarily directed to usingangiotensin-converting enzyme (ACE) inhibitors, such as captopril, anddiuretics. These drugs improve hemodynamic profile and exercisetolerance and reduce the incidence of morbidity and mortality inpatients with CHF. Kramer et al., Circulation, 67(4): 807–816 (1983);Captopril Multicenter Research Group, J.A.C.C., 2(4): 755–763 (1983);The CONSENSUS Trial Study Group, N. Engl. J. Med., 316(23): 1429–1435(1987); The SOLVD Investigators, N. Engl. J. Med., 325(5): 293–302(1991). Further, they are useful in treating hypertension, leftventricular dysfunction, atherosclerotic vascular disease, and diabeticnephropathy. Brown and Vaughan, supra. However, despite proven efficacy,response to ACE inhibitors has been limited. For example, whileprolonging survival in the setting of heart failure, ACE inhibitorsappear to slow the progression towards end-stage heart failure, andsubstantial numbers of patients on ACE inhibitors have functional classIII heart failure.

Moreover, improvement of functional capacity and exercise time is onlysmall and mortality, although reduced, continues to be high. TheCONSENSUS Trial Study Group, N. Engl. J. Med., 316(23): 1429–1453(1987); The SOLVD Investigators, N. Engl. J. Med., 325(5): 293–302(1991); Cohn et al., N. Engl. J. Med., 325(5): 303–310 (1991); TheCaptopril-Digoxin Multicenter Research Group, JAMA, 259(4): 539–544(1988). Hence, ACE inhibitors consistently appear unable to relievesymptoms in more than 60% of heart failure patients and reduce mortalityof heart failure only by approximately 15–20%. For further adverseeffects, see Brown and Vaughan, supra.

An alternative to ACE inhibitors is represented by specific AT1 receptorantagonists. Clinical studies are planned to compare the efficacy ofthese two modalities in the treatment of cardiovascular and renaldisease. However, animal model data suggests that the ACE/Ang IIpathway, while clearly involved in cardiac hypertrophy, is not the only,or even the primary pathway active in this role. Mouse genetic“knockout” models have been made to test individual components of thepathway. In one such model, the primary cardiac receptor for Ang II, ATsub 1A, has been genetically deleted; these mice do not develophypertrophy when Ang II is given experimentally (confirming the basicsuccess of the model in eliminating hypertrophy secondary to Ang II).However, when the aorta is constricted in these animals (a model ofhypertensive cardiac stress), the hearts still become hypertrophic. Thissuggests that alternative signaling pathways, not depending on thisreceptor (AT sub 1A), are activated in hypertension. ACE inhibitorswould presumably not be able to inhibit these pathways. See, Harada etal., Circulation, 97: 1952–1959 (1998). See also, Homcy, Circulation,97: 1890–1892 (1998) regarding the enigma associated with the processand mechanism of cardiac hypertrophy.

About 750,000 patients suffer from acute myocardial infarction (AMI)annually, and approximately one-fourth of all deaths in the UnitedStates are due to AMI. In recent years, thrombolytic agents, e.g.,streptokinase, urokinase, and in particular tissue plasminogen activator(t-PA) have significantly increased the survival of patients whosuffered myocardial infarction. When administered as a continuousintravenous infusion over 1.5 to 4 hours, t-PA produces coronary patencyat 90 minutes in 69% to 90% of the treated patients. Topol et al., Am.J. Cardiol., 61: 723–728 (1988); Neuhaus et al., J. Am. Coll. Cardiol.,12: 581–587 (1988); Neuhaus et al., J. Am. Coll. Cardiol., 14: 1566–1569(1989). The highest patency rates have been reported with high dose oraccelerated dosing regimens. Topol, J. Am. Coll. Cardiol., 15: 922–924(1990). t-PA may also be administered as a single bolus, although due toits relatively short half-life, it is better suited for infusiontherapy. Tebbe et al., Am. J. Cardiol., 64: 448–453 (1989). At-PAvariant, specifically designed to have longer half-life and very highfibrin specificity, TNK t-PA (a T103N, N117Q, KHRR(296–299)AAAA t-PAvariant, Keyt et al., Proc. Natl. Acad. Sci. USA, 91: 3670–3674(1994))is particularly suitable for bolus administration. However, despite allthese advances, the long-term prognosis of patient survival dependsgreatly on the post-infarction monitoring and treatment of the patients,which should include monitoring and treatment of cardiac hypertrophy.

2.3. Growth Factors

Various naturally occurring polypeptides reportedly induce theproliferation of endothelial cells. Among those polypeptides are thebasic and acidic fibroblast growth factors (FGF) (Burgess and Maciag,Annual Rev. Biochem., 58: 575 (1989)), platelet-derived endothelial cellgrowth factor (PD-ECGF) (Ishikawa et al., Nature, 338: 557 (1989)), andvascular endothelial growth factor (VEGF). Leung et al., Science, 246:1306 (1989); Ferrara and Henzel, Biochem. Biophys. Res. Commun., 161:851 (1989); Tischer et al., Biochem. Biophys. Res. Commun., 165: 1198(1989); EP 471,754B granted Jul. 31, 1996.

Media conditioned by cells transfected with the human VEGF (hVEGF) cDNApromoted the proliferation of capillary endothelial cells, whereascontrol cells did not. Leung et al., Science, 246: 1306 (1989). Severaladditional cDNAs were identified in human cDNA libraries that encode121-, 189-, and 206-amino acid isoforms of hVEGF (also collectivelyreferred to as hVEGF-related proteins). The 121-amino acid proteindiffers from hVEGF by virtue of the deletion of the 44 amino acidsbetween residues 116 and 159 in hVEGF. The 189-amino acid proteindiffers from hVEGF by virtue of the insertion of 24 amino acids atresidue 116 in hVEGF, and apparently is identical to human vascularpermeability factor (hVPF). The 206-amino acid protein differs fromhVEGF by virtue of an insertion of 41 amino acids at residue 116 inhVEGF. Houck et al., Mol. Endocrin., 5: 1806 (1991); Ferrara et al., J.Cell. Biochem., 47: 211 (1991); Ferrara et al., Endocrine Reviews, 13:18 (1992); Keck et al., Science, 246: 1309 (1989); Connolly et al., J.Biol. Chem., 264: 20017 (1989); EP 370,989 published May 30, 1990.

It is now well established that angiogenesis, which involves theformation of new blood vessels from preexisting endothelium, isimplicated in the pathogenesis of a variety of disorders. These includesolid tumors and metastasis, atherosclerosis, retrolental fibroplasia,hemangiomas, chronic inflammation, intraocular neovascular syndromessuch as proliferative retinopathies, e.g., diabetic retinopathy,age-related macular degeneration (AMD), neovascular glaucoma, immunerejection of transplanted corneal tissue and other tissues, rheumatoidarthritis, and psoriasis. Folkman et al., J. Biol. Chem., 267:10931–10934 (1992); Klagsbrun et al., Annu. Rev. Physiol., 53: 217–239(1991); and Garner A., “Vascular diseases”, In: Pathobiology of OcularDisease. A Dynamic Approach, Garner A., Klintworth G K, eds., 2ndEdition (Marcel Dekker, NY, 1994), pp 1625–1710.

In the case of tumor growth, angiogenesis appears to be crucial for thetransition from hyperplasia to neoplasia, and for providing nourishmentfor the growth and metastasis of the tumor. Folkman et al., Nature, 339:58 (1989). The neovascularization allows the tumor cells to acquire agrowth advantage and proliferative autonomy compared to the normalcells. A tumor usually begins as a single aberrant cell which canproliferate only to a size of a few cubic millimeters due to thedistance from available capillary beds, and it can stay ‘dormant’without further growth and dissemination for a long period of time. Sometumor cells then switch to the angiogenic phenotype to activateendothelial cells, which proliferate and mature into new capillary bloodvessels. These newly formed blood vessels not only allow for continuedgrowth of the primary tumor, but also for the dissemination andrecolonization of metastatic tumor cells. Accordingly, a correlation hasbeen observed between density of microvessels in tumor sections andpatient survival in breast cancer as well as in several other tumors.Weidner et al., N. Engl. J. Med, 324: 1–6 (1991); Horak et al., Lancet,340: 1120–1124 (1992); Macchiarini et al., Lancet, 340: 145–146 (1992).The precise mechanisms that control the angiogenic switch is not wellunderstood, but it is believed that neovascularization of tumor massresults from the net balance of a multitude of angiogenesis stimulatorsand inhibitors (Folkman, 1995, Nat Med 1(1):27–31).

The search for positive regulators of angiogenesis has yielded manycandidates, including aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α, angiogenin,IL-8, etc. Folkman et al., J.B.C., supra, and Klagsbrun et al., supra.The negative regulators so far identified include thrombospondin (Goodet al., Proc. Natl. Acad. Sci. USA., 87: 6624–6628 (1990)), the16-kilodalton N-terminal fragment of prolactin (Clapp et al.,Endocrinology, 133: 1292–1299 (1993)), angiostatin (O'Reilly et al.,Cell, 79: 315–328 (1994)), and endostatin. O'Reilly et al., Cell, 88:277–285 (1996).

Work done over the last several years has established the key role ofVEGF, not only in stimulating vascular endothelial cell proliferation,but also in inducing vascular permeability and angiogenesis. Ferrara etal., Endocr. Rev., 18: 4–25 (1997). The finding that the loss of even asingle VEGF allele results in embryonic lethality points to anirreplaceable role played by this factor in the development anddifferentiation of the vascular system. Furthermore, VEGF has been shownto be a key mediator of neovascularization associated with tumors andintraocular disorders. Ferrara et al., Endocr. Rev., supra. The VEGFmRNA is overexpressed by the majority of human tumors examined. Berkmanet al., J. Clin. Invest., 91: 153–159 (1993); Brown et al., HumanPathol., 26: 86–91 (1995); Brown et al., Cancer Res., 53: 4727–4735(1993); Mattern et al., Brit. J. Cancer, 73: 931–934 (1996); Dvorak etal., Am. J. Pathol., 146: 1029–1039 (1995).

Also, the concentration levels of VEGF in eye fluids are highlycorrelated to the presence of active proliferation of blood vessels inpatients with diabetic and other ischemia-related retinopathies. Aielloet al., N. Engl. J. Med., 331: 1480–1487 (1994). Furthermore, recentstudies have demonstrated the localization of VEGF in choroidalneovascular membranes in patients affected by AMD. Lopez et al., Invest.Ophthalmol. Vis. Sci., 37: 855–868 (1996).

Anti-VEGF neutralizing antibodies suppress the growth of a variety ofhuman tumor cell lines in nude mice (Kim et al., Nature, 362: 841–844(1993); Warren et al., J. Clin. Invest., 95: 1789–1797 (1995); Borgströmet al., Cancer Res., 56: 4032–4039 (1996); Melnyk et al., Cancer Res.,56: 921–924 (1996)) and also inhibit intraocular angiogenesis in modelsof ischemic retinal disorders. Adamis et al., Arch. Ophthalmol., 114:66–71 (1996). Therefore, anti-VEGF monoclonal antibodies or otherinhibitors of VEGF action are promising candidates for the treatment ofsolid tumors and various intraocular neovascular disorders. Suchantibodies are described, for example, in EP 817,648 published Jan. 14,1998 and in WO98/45331 and WO98/45332 both published Oct. 15, 1998.

There exist several other growth factors and mitogens, includingtransforming oncogenes, that are capable of rapidly inducing a complexset of genes to be expressed by certain cells. Lau and Nathans,Molecular Aspects of Cellular Regulation, 6: 165–202 (1991). Thesegenes, which have been named immediate-early- or early-response genes,are transcriptionally activated within minutes after contact with agrowth factor or mitogen, independent of de novo protein synthesis. Agroup of these intermediate-early genes encodes secreted, extracellularproteins that are needed for coordination of complex biologicalprocesses such as differentiation and proliferation, regeneration, andwound healing. Ryseck et al., Cell Growth Differ., 2: 235–233 (1991).

Highly-related proteins that belong to this group include cef 10(Simmons et al., Proc. Natl. Acad. Sci. USA, 86: 1178–1182 (1989)), cyr61, which is rapidly activated by serum- or platelet-derived growthfactor (PDGF) (O'Brien et al., Mol. Cell Biol., 10: 3569–3577 (1990),human connective tissue growth factor (CTGF) (Bradham et al., J. Cell.Biol., 114: 1285–1294 (1991)), which is secreted by human vascularendothelial cells in high levels after activation with transforminggrowth factor beta (TGF-β), exhibits PDGF-like biological andimmunological activities, and competes with PDGF for a particular cellsurface receptor, fisp-12 (Ryseck et al., Cell Growth Differ., 2:235–233 (1991)), human vascular IBP-like growth factor (VIGF) (WO96/17931), and nov, normally arrested in adult kidney cells, which wasfound to be overexpressed inmyeloblastosis-associated-virus-type-1-induced nephroblastomas. Joloitet al., Mol. Cell. Biol., 12: 10–21 (1992).

The expression of these immediate-early genes acts as “third messengers”in the cascade of events triggered by growth factors. It is also thoughtthat they are needed to integrate and coordinate complex biologicalprocesses, such as differentiation and wound healing in which cellproliferation is a common event.

As additional mitogens, insulin-like growth factor binding proteins(IGFBPs) have been shown, in complex with insulin-like growth factor(IGF), to stimulate increased binding of IGF to fibroblast and smoothmuscle cell surface receptors. Clemmons et al., J. Clin. Invest., 77:1548 (1986). Inhibitory effects of IGFBP on various IGF actions in vitroinclude stimulation of glucose transport by adipocytes, sulfateincorporation by chondrocytes, and thymidine incorporation infibroblast. Zapf et al., J. Clin. Invest., 63: 1077 (1979). In addition,inhibitory effects of IGFBPs on growth factor-mediated mitogen activityin normal cells have been shown.

2.4. Need for Further Treatments

In view of the role of vascular endothelial cell growth and angiogenesisin many diseases and disorders, it is desirable to have a means ofreducing or inhibiting one or more of the biological effects causingthese processes. It is also desirable to have a means of assaying forthe presence of pathogenic polypeptides in normal and diseasedconditions, and especially cancer. Further, in a specific aspect, asthere is no generally applicable therapy for the treatment of cardiachypertrophy, the identification of factors that can prevent or reducecardiac myocyte hypertrophy is of primary importance in the developmentof new therapeutic strategies to inhibit pathophysiological cardiacgrowth. While there are several treatment modalities for variouscardiovascular and oncologic disorders, there is still a need foradditional therapeutic approaches.

3. SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulating(e.g., promoting or inhibiting) angiogenesis and/orcardiovascularization in mammals. The present invention is based on theidentification of compounds (i.e., proteins) that test positive invarious cardiovascular assays that test modulation (e.g., promotion orinhibition) of certain biological activities. Accordingly, the compoundsare believed to be useful drugs and/or drug components for the diagnosisand/or treatment (including prevention and amelioration) of disorderswhere such effects are desired, such as the promotion or inhibition ofangiogenesis, inhibition or stimulation of vascular endothelial cellgrowth, stimulation of growth or proliferation of vascular endothelialcells, inhibition of tumor growth, inhibition of angiogenesis-dependenttissue growth, stimulation of angiogenesis-dependent tissue growth,inhibition of cardiac hypertrophy and stimulation of cardiachypertrophy, e.g., for the treatment of congestive heart failure. Inaddition, the compositions and methods of the invention provide for thediagnostic monitoring of patients undergoing clinical evaluation for thetreatment of angiogenesis-related disorders, for monitoring the efficacyof compounds in clinical trials and for identifying subjects who may bepredisposed to such angiogenic-related disorders.

In one embodiment, the present invention provides a compositioncomprising a PRO polypeptide, an agonist or antagonist thereof, or ananti-PRO antibody in admixture with a pharmaceutically acceptablecarrier. In one aspect, the composition comprises a therapeuticallyeffective amount of the polypeptide, agonist, antagonist or antibody. Inanother aspect, the composition comprises a further active ingredient,namely, a cardiovascular, endothelial or angiogenic agent or anangiostatic agent, preferably an angiogenic or angiostatic agent.Preferably, the composition is sterile. The PRO polypeptide, agonist,antagonist or antibody may be administered in the form of a liquidpharmaceutical formulation, which may be preserved to achieve extendedstorage stability. Preserved liquid pharmaceutical formulations mightcontain multiple doses of PRO polypeptide, agonist, antagonist orantibody, and might, therefore, be suitable for repeated use. In apreferred embodiment, where the composition comprises an antibody, theantibody is a monoclonal antibody, an antibody fragment, a humanizedantibody, or a single-chain antibody.

In a further embodiment, the present invention provides a method forpreparing such a composition useful for the treatment of acardiovascular, endothelial or angiogenic disorder comprising admixing atherapeutically effective amount of a PRO polypeptide, agonist,antagonist or antibody with a pharmaceutically acceptable carrier.

In a still further aspect, the present invention provides an article ofmanufacture comprising:

(a) a composition of matter comprising a PRO polypeptide or agonist orantagonist thereof;

(b) a container containing said composition; and

(c) a label affixed to said container, or a package insert included insaid container referring to the use of said PRO polypeptide or agonistor antagonist thereof in the treatment of a cardiovascular, endothelialor angiogenic disorder, wherein the agonist or antagonist may be anantibody which binds to the PRO polypeptide. The composition maycomprise a therapeutically effective amount of the PRO polypeptide orthe agonist or antagonist thereof.

In another embodiment, the present invention provides a method foridentifying an agonist of a PRO polypeptide comprising:

(a) contacting cells and a test compound to be screened under conditionssuitable for the induction of a cellular response normally induced by aPRO polypeptide; and

(b) determining the induction of said cellular response to determine ifthe test compound is an effective agonist, wherein the induction of saidcellular response is indicative of said test compound being an effectiveagonist.

In another embodiment, the present invention provides a method foridentifying an agonist of a PRO polypeptide comprising:

(a) contacting cells and a test compound to be screened under conditionssuitable for the stimulation of cell proliferation by a PRO polypeptide;and

(b) measuring the proliferation of said cells to determine if the testcompound is an effective agonist, wherein the stimulation of cellproliferation is indicative of said test compound being an effectiveagonist.

In another embodiment, the invention provides a method for identifying acompound that inhibits the activity of a PRO polypeptide comprisingcontacting a test compound with a PRO polypeptide under conditions andfor a time sufficient to allow the test compound and polypeptide tointeract and determining whether the activity of the PRO polypeptide isinhibited. In a specific preferred aspect, either the test compound orthe PRO polypeptide is immobilized on a solid support. In anotherpreferred aspect, the non-immobilized component carries a detectablelabel. In a preferred aspect, this method comprises the steps of:

(a) contacting cells and a test compound to be screened in the presenceof a PRO polypeptide under conditions suitable for the induction of acellular response normally induced by a PRO polypeptide; and

(b) determining the induction of said cellular response to determine ifthe test compound is an effective antagonist.

In another preferred aspect, this process comprises the steps of:

(a) contacting cells and a test compound to be screened in the presenceof a PRO polypeptide under conditions suitable for the stimulation ofcell proliferation by a PRO polypeptide; and

(b) measuring the proliferation of the cells to determine if the testcompound is an effective antagonist.

In another embodiment, the invention provides a method for identifying acompound that inhibits the expression of a PRO polypeptide in cells thatnormally expresses the polypeptide, wherein the method comprisescontacting the cells with a test compound and determining whether theexpression of the PRO polypeptide is inhibited. In a preferred aspect,this method comprises the steps of:

(a) contacting cells and a test compound to be screened under conditionssuitable for allowing expression of the PRO polypeptide; and

(b) determining the inhibition of expression of said polypeptide.

In a still further embodiment, the invention provides a compound thatinhibits the expression of a PRO polypeptide, such as a compound that isidentified by the methods set forth above.

Another aspect of the present invention is directed to an agonist or anantagonist of a PRO polypeptide which may optionally be identified bythe methods described above.

One type of antagonist of a PRO polypeptide that inhibits one or more ofthe functions or activities of the PRO polypeptide is an antibody.Hence, in another aspect, the invention provides an isolated antibodythat binds a PRO polypeptide. In a preferred aspect, the antibody is amonoclonal antibody, which preferably has non-humancomplementarity-determining-region (CDR) residues and humanframework-region (FR) residues. The antibody may be labeled and may beimmobilized on a solid support. In a further aspect, the antibody is anantibody fragment, a single-chain antibody, or a humanized antibody.Preferably, the antibody specifically binds to the polypeptide.

In a still further aspect, the present invention provides a method fordiagnosing a disease or susceptibility to a disease which is related toa mutation in a PRO polypeptide-encoding nucleic acid sequencecomprising determining the presence or absence of said mutation in thePRO polypeptide nucleic acid sequence, wherein the presence or absenceof said mutation is indicative of the presence of said disease orsusceptibility to said disease.

In a still further aspect, the invention provides a method of diagnosinga cardiovascular, endothelial or angiogenic disorder in a mammal whichcomprises analyzing the level of expression of a gene encoding a PROpolypeptide (a) in a test sample of tissue cells obtained from saidmammal, and (b) in a control sample of known normal tissue cells of thesame cell type, wherein a higher or lower expression level in the testsample as compared to the control sample is indicative of the presenceof a cardiovascular, endothelial or angiogenic disorder in said mammal.The expression of a gene encoding a PRO polypeptide may optionally beaccomplished by measuring the level of mRNA or the polypeptide in thetest sample as compared to the control sample.

In a still further aspect, the present invention provides a method ofdiagnosing a cardiovascular, endothelial or angiogenic disorder in amammal which comprises detecting the presence or absence of a PROpolypeptide in a test sample of tissue cells obtained from said mammal,wherein the presence or absence of said PRO polypeptide in said testsample is indicative of the presence of a cardiovascular, endothelial orangiogenic disorder in said mammal.

In a still further embodiment, the invention provides a method ofdiagnosing a cardiovascular, endothelial or angiogenic disorder in amammal comprising (a) contacting an anti-PRO antibody with a test sampleof tissue cells obtained from the mammal, and (b) detecting theformation of a complex between the antibody and the PRO polypeptide inthe test sample, wherein the formation of said complex is indicative ofthe presence of a cardiovascular, endothelial or angiogenic disorder inthe mammal. The detection may be qualitative or quantitative, and may beperformed in comparison with monitoring the complex formation in acontrol sample of known normal tissue cells of the same cell type. Alarger or smaller quantity of complexes formed in the test sampleindicates the presence of a cardiovascular, endothelial or angiogenicdysfunction in the mammal from which the test tissue cells wereobtained. The antibody preferably carries a detectable label. Complexformation can be monitored, for example, by light microscopy, flowcytometry, fluorimetry, or other techniques known in the art. The testsample is usually obtained from an individual suspected to have acardiovascular, endothelial or angiogenic disorder.

In another embodiment, the invention provides a method for determiningthe presence of a PRO polypeptide in a sample comprising exposing asample suspected of containing the PRO polypeptide to an anti-PROantibody and determining binding of said antibody to a component of saidsample. In a specific aspect, the sample comprises a cell suspected ofcontaining the PRO polypeptide and the antibody binds to the cell. Theantibody is preferably detectably labeled and/or bound to a solidsupport.

In further aspects, the invention provides a cardiovascular, endothelialor angiogenic disorder diagnostic kit comprising an anti-PRO antibodyand a carrier in suitable packaging. Preferably, such kit furthercomprises instructions for using said antibody to detect the presence ofthe PRO polypeptide. Preferably, the carrier is a buffer, for example.Preferably, the cardiovascular, endothelial or angiogenic disorder iscancer.

In yet another embodiment, the present invention provides a method fortreating a cardiovascular, endothelial or angiogenic disorder in amammal comprising administering to the mammal an effective amount of aPRO polypeptide. Preferably, the disorder is cardiac hypertrophy, traumasuch as wounds or burns, or a type of cancer. In a further aspect, themammal is further exposed to angioplasty or a drug that treatscardiovascular, endothelial or angiogenic disorders such as ACEinhibitors or chemotherapeutic agents if the cardiovascular, endothelialor angiogenic disorder is a type of cancer. Preferably, the mammal ishuman, preferably one who is at risk of developing cardiac hypertrophyand more preferably has suffered myocardial infarction.

In another preferred aspect, the cardiac hypertrophy is characterized bythe presence of an elevated level of PGF_(2α). Alternatively, thecardiac hypertrophy may be induced by myocardial infarction, whereinpreferably the administration of the PRO polypeptide is initiated within48 hours, more preferably within 24 hours, following myocardialinfarction.

In another preferred embodiment, the cardiovascular, endothelial orangiogenic disorder is cardiac hypertrophy and said PRO polypeptide isadministered together with a cardiovascular, endothelial or angiogenicagent. The preferred cardiovascular, endothelial or angiogenic agent forthis purpose is selected from the group consisting of anantihypertensive drug, an ACE inhibitor, an endothelin receptorantagonist and a thrombolytic agent. If a thrombolytic agent isadministered, preferably the PRO polypeptide is administered followingadministration of such agent. More preferably, the thrombolytic agent isrecombinant human tissue plasminogen activator.

In another preferred aspect, the cardiovascular, endothelial orangiogenic disorder is cardiac hypertrophy and the PRO polypeptide isadministered following primary angioplasty for the treatment of acutemyocardial infarction, preferably wherein the mammal is further exposedto angioplasty or a cardiovascular, endothelial, or angiogenic agent.

In another preferred embodiment, the cardiovascular, endothelial orangiogenic disorder is a cancer and the PRO polypeptide is administeredin combination with a chemotherapeutic agent, a growth inhibitory agentor a cytotoxic agent.

In a further embodiment, the invention provides a method for treating acardiovascular, endothelial or angiogenic disorder in a mammalcomprising administering to the mammal an effective amount of a PROpolypeptide agonist, antagonist or anti-PRO antibody. Preferably, thecardiovascular, endothelial or angiogenic disorder is cardiachypertrophy, trauma, a cancer, or age-related macular degeneration. Alsopreferred is where the mammal is human, and where an effective amount ofan angiogenic or angiostatic agent is administered in conjunction withthe agonist, antagonist or anti-PRO antibody.

In still further embodiments, the invention provides a method fortreating a cardiovascular, endothelial or angiogenic disorder in amammal that suffers therefrom comprising administering to the mammal anucleic acid molecule that codes for either (a) a PRO polypeptide, (b)an agonist of a PRO polypeptide or (c) an antagonist of a PROpolypeptide, wherein said agonist or antagonist may be an anti-PROantibody. In a preferred embodiment, the mammal is human. In anotherpreferred embodiment, the gene is administered via ex vivo gene therapy.In a further preferred embodiment, the gene is comprised within avector, more preferably an adenoviral, adeno-associated viral,lentiviral, or retroviral vector.

In yet another aspect, the invention provides a recombinant retroviralparticle comprising a retroviral vector consisting essentially of apromoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonistpolypeptide of a PRO polypeptide, or (c) an antagonist polypeptide of aPRO polypeptide, and a signal sequence for cellular secretion of thepolypeptide, wherein the retroviral vector is in association withretroviral structural proteins. Preferably, the signal sequence is froma mammal, such as from a native PRO polypeptide.

In a still further embodiment, the invention supplies an ex vivoproducer cell comprising a nucleic acid construct that expressesretroviral structural proteins and also comprises a retroviral vectorconsisting essentially of a promoter, nucleic acid encoding (a) a PROpolypeptide, (b) an agonist polypeptide of a PRO polypeptide or (c) anantagonist polypeptide of a PRO polypeptide, and a signal sequence forcellular secretion of the polypeptide, wherein said producer cellpackages the retroviral vector in association with the structuralproteins to produce recombinant retroviral particles.

In yet another embodiment, the invention provides a method forinhibiting endothelial cell growth in a mammal comprising administeringto the mammal (a) a PRO polypeptide, (b) an agonist of a PROpolypeptide, or (c) an antagonist of a PRO polypeptide, whereinendothelial cell growth in said mammal is inhibited, and wherein saidagonist or antagonist may be an anti-PRO antibody. Preferably, themammal is human and the endothelial cell growth is associated with atumor or a retinal disorder.

In yet another embodiment, the invention provides a method forstimulating endothelial cell growth in a mammal comprising administeringto the mammal (a) a PRO polypeptide, (b) an agonist of a PROpolypeptide, or (c) an antagonist of a PRO polypeptide, whereinendothelial cell growth in said mammal is stimulated, and wherein saidagonist or antagonist may be an anti-PRO antibody. Preferably, themammal is human.

In yet another embodiment, the invention provides a method forinhibiting cardiac hypertrophy in a mammal comprising administering tothe mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide,or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophyin said mammal is inhibited, and wherein said agonist or antagonist maybe an anti-PRO antibody. Preferably, the mammal is human and the cardiachypertrophy has been induced by myocardial infarction.

In yet another embodiment, the invention provides a method forstimulating cardiac hypertrophy in a mammal comprising administering tothe mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide,or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophyin said mammal is stimulated, and wherein said agonist or antagonist maybe an anti-PRO antibody. Preferably, the mammal is human who suffersfrom congestive heart failure.

In yet another embodiment, the invention provides a method forinhibiting angiogenesis induced by a PRO polypeptide in a mammalcomprising administering a therapeutically effective amount of ananti-PRO antibody to the mammal. Preferably, the mammal is a human, andmore preferably the mammal has a tumor or a retinal disorder.

In yet another embodiment, the invention provides a method forstimulating angiogenesis induced by a PRO polypeptide in a mammalcomprising administering a therapeutically effective amount of a PROpolypeptide to the mammal. Preferably, the mammal is a human, and morepreferably angiogenesis would promote tissue regeneration or woundhealing.

In yet another embodiment, the invention provides a method formodulating (e.g., inhibiting or stimulating) endothelial cell growth ina mammal comprising administering to the mammal a PRO21, PRO 181,PRO205, PRO214, PRO221, PRO229, PRO231, PRO238, PRO241, PRO247, PRO256,PRO258, PRO263, PRO265, PRO295, PRO321, PRO322, PRO337, PRO363, PRO365,PRO444, PRO533, PRO697, PRO720, PRO725, PRO771, PRO788, PRO791, PRO819,PRO827, PRO828, PRO836, PRO846, PRO865, PRO1005, PRO1006, PRO1007,PRO1025, PRO1029, PRO1054, PRO1071, PRO1075, PRO1079, PRO1080, PRO1114,PRO1131, PRO1155, PRO1160, PRO1184, PRO1186, PRO1190, PRO1192, PRO1195,PRO1244, PRO1272, PRO1273, PRO1274, PRO1279, PRO1283, PRO1286, PRO1306,PRO1309, PRO1325, PRO1329, PRO1347, PRO1356, PRO1376, PRO1382, PRO1411,PRO1412, PRO1419, PRO1474, PRO1477, PRO1488, PRO1508, PRO1550, PRO1556,PRO1760, PRO1782, PRO1787, PRO1801, PRO1868, PRO1887, PRO1890, PRO3438,PRO3444, PRO4302, PRO4324, PRO4333, PRO4341, PRO4342, PRO4353, PRO4354,PRO4356, PRO4371, PRO4405, PRO4408, PRO4422, PRO4425, PRO4499, PRO5723,PRO5725, PRO5737, PRO5776, PRO6006, PRO6029, PRO6071, PRO7436, PRO9771,PRO9821, PRO9873, PRO10008, PRO10096, PRO19670, PRO20040, PRO20044,PRO21055, PRO21384 or PRO28631 polypeptide, agonist or antagonistthereof, wherein endothelial cell growth in said mammal is modulated.

In yet another embodiment, the invention provides a method formodulating (e.g., inhibiting or stimulating) smooth muscle cell growthin a mammal comprising administering to the mammal a PRO162, PRO181,PRO182, PRO195, PRO204, PRO221, PRO230, PRO256, PRO258, PRO533, PRO697,PRO725, PRO738, PRO826, PRO836,PRO840, PRO846,PRO865, PRO982, PRO1025,PRO1029, PRO1071, PRO1080, PRO1083, PRO1134, PRO1160, PRO1182, PRO1184,PRO1186, PRO1192, PRO1265, PRO1274, PRO1279, PRO1283, PRO1306, PRO1308,PRO1309, PRO1325, PRO1337, PRO1338, PRO1343, PRO1376, PRO1387, PRO1411,PRO1412, PRO1415, PRO1434, PRO1474, PRO1488, PRO1550, PRO1556, PRO1567,PRO1600, PRO1754, PRO1758, PRO1760, PRO1787, PRO1865, PRO1868, PRO1917,PRO1928, PRO3438, PRO3562, PRO4302, PRO4333, PRO4345, PRO4353, PRO4354,PRO4405, PRO4408, PRO4430, PRO4503, PRO5725, PRO6714, PRO9771, PRO9820,PRO9940, PRO10096, PRO21055, PRO21184 or PRO21366 polypeptide, agonistor antagonist thereof, wherein endothelial cell growth in said mammal ismodulated.

In yet another embodiment, the invention provides a method formodulating (e.g., inducing or reducing) cardiac hypertrophy in a mammalcomprising administering to the mammal a PRO21 polypeptide, agonist orantagonist thereof, wherein cardiac hypertrophy in said mammal ismodulated.

In yet another embodiment, the invention provides a method formodulating (e.g., inducing or reducing) endothelial cell apoptosis in amammal comprising administering to the mammal a PRO4302 polypeptide,agonist or antagonist thereof, wherein cardiac hypertrophy in saidmammal is modulated.

In yet another embodiment, the invention provides a method formodulating (e.g., stimulating or inhibiting) angiogenesis in a mammalcomprising administering a therapeutically effective amount of a PRO1376or PRO1449 polypeptide, agonist or antagonist thereof to the mammal,wherein said angiogenesis is modulated.

In yet another embodiment, the invention provides a method formodulating (e.g., inducing or reducing) angiogenesis by modulating(e.g., inducing or reducing) endothelial cell tube formation in a mammalcomprising administering to the mammal a PRO178, PRO195, PRO228, PRO301,PRO302, PRO532, PRO724, PRO730, PRO734, PRO793, PRO871,PRO938, PRO1012,PRO1120,PRO1139,PRO1198, PRO1287, PRO1361, PRO1864, PRO1873, PRO2010,PRO3579, PRO4313, PRO4527, PRO4538, PRO4553, PRO4995, PRO5730, PRO6008,PRO7223, PRO7248 or PRO7261 polypeptide, agonist or antagonist thereof,wherein endothelial cell tube formation in said mammal is modulated.

In other embodiments of the present invention, the invention provides anisolated nucleic acid molecule comprising a nucleotide sequence thatencodes a PRO polypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) a DNA molecule encoding a PRO polypeptidehaving a full-length amino acid sequence as disclosed herein, an aminoacid sequence lacking the signal peptide as disclosed herein, anextracellular domain of a transmembrane protein, with or without thesignal peptide, as disclosed herein or any other specifically definedfragment of the full-length amino acid sequence as disclosed herein, or(b) the complement of the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%nucleic acid sequence identity and alternatively at least about 99%nucleic acid sequence identity to (a) a DNA molecule comprising thecoding sequence of a full-length PRO polypeptide cDNA as disclosedherein, the coding sequence of a PRO polypeptide lacking the signalpeptide as disclosed herein, the coding sequence of an extracellulardomain of a transmembrane PRO polypeptide, with or without the signalpeptide, as disclosed herein or the coding sequence of any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein, or (b) the complement of the DNA molecule of (a).

In a further aspect, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% or 98% nucleic acid sequence identity and alternatively atleast about 99% nucleic acid sequence identity to (a) a DNA moleculethat encodes the same mature polypeptide encoded by any of the humanprotein cDNAs deposited with the ATCC as disclosed herein, or (b) thecomplement of the DNA molecule of (a).

Another aspect of the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence encoding a PROpolypeptide which is either transmembrane domain-deleted ortransmembrane domain-inactivated, or is complementary to such encodingnucleotide sequence, wherein the transmembrane domain(s) of suchpolypeptide are disclosed herein. Therefore, soluble extracellulardomains of the herein described PRO polypeptides are contemplated.

Another embodiment is directed to fragments of a PRO polypeptide codingsequence, or the complement thereof, that may find use as, for example,hybridization probes, for encoding fragments of a PRO polypeptide thatmay optionally encode a polypeptide comprising a binding site for ananti-PRO antibody or as antisense oligonucleotide probes. Such nucleicacid fragments are usually at least about 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, 600, 700 or 800 nucleotides in length andalternatively at least about 1000 nucleotides in length, wherein in thiscontext the term “about” means the referenced nucleotide sequence lengthplus or minus 10% of that referenced length. It is noted that novelfragments of a PRO polypeptide-encoding nucleotide sequence may bedetermined in a routine manner by aligning the PRO polypeptide-encodingnucleotide sequence with other known nucleotide sequences using any of anumber of well known sequence alignment programs and determining whichPRO polypeptide-encoding nucleotide sequence fragment(s) are novel. Allof such PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

In another embodiment, the invention provides an isolated PROpolypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

In a certain aspect, the invention provides an isolated PRO polypeptidecomprising an amino acid sequence having at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97% or 98% amino acid sequence identity and alternatively at least about99% amino acid sequence identity to a PRO polypeptide having afull-length amino acid sequence as disclosed herein, an amino acidsequence lacking the signal peptide as disclosed herein, anextracellular domain of a transmembrane protein, with or without thesignal peptide, as disclosed herein or any other specifically definedfragment of the full-length amino acid sequence as disclosed herein.

In a further aspect, the invention provides an isolated PRO polypeptidecomprising an amino acid sequence having at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97% or 98% amino acid sequence identity and alternatively at least about99% amino acid sequence identity to an amino acid sequence encoded byany of the human protein cDNAs deposited with the ATCC as disclosedherein.

In a specific aspect, the invention provides an isolated PRO polypeptidewithout the N-terminal signal sequence and/or the initiating methionineand that is encoded by a nucleotide sequence that encodes such an aminoacid sequence as hereinbefore described. Processes for producing thesame are also herein described, wherein those processes compriseculturing a host cell comprising a vector which comprises theappropriate encoding nucleic acid molecule under conditions suitable forexpression of the PRO polypeptide and recovering the PRO polypeptidefrom the cell culture.

Another aspect of the invention provides an isolated PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

In yet another embodiment, the invention provides agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

In a further embodiment, the invention provides a method of identifyingagonists or antagonists to a PRO polypeptide which comprise contactingthe PRO polypeptide with a candidate molecule and monitoring abiological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

In a still further embodiment, the invention provides a composition ofmatter comprising a PRO polypeptide, or an agonist or antagonist of aPRO polypeptide as herein described, or an anti-PRO antibody, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO polypeptide, or an agonist or antagonist thereof as hereinbeforedescribed, or an anti-PRO antibody, for the preparation of a medicamentuseful in the treatment of a condition which is responsive to the PROpolypeptide, an agonist or antagonist thereof or an anti-PRO antibody.

In additional embodiments of the present invention, the inventionprovides vectors comprising DNA encoding any of the herein describedpolypeptides. Host cells comprising any such vector are also provided.By way of example, the host cells may be CHO cells, E. coli, yeast, orBaculovirus-infected insect cells. A process for producing any of theherein described polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of thedesired polypeptide and recovering the desired polypeptide from the cellculture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In yet another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO181 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA23330-1390”.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequencePRO178 cDNA, wherein SEQ ID NO:3 is a clone designated herein as“DNA23339-1130”.

FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived from thecoding sequence of SEQ ID NO:3 shown in FIG. 3.

FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a native sequencePRO444 cDNA, wherein SEQ ID NO:5 is a clone designated herein as“DNA26846-1397”.

FIG. 6 shows the amino acid sequence (SEQ ID NO:6) derived from thecoding sequence of SEQ ID NO:5 shown in FIG. 5.

FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a native sequencePRO195 cDNA, wherein SEQ ID NO:7 is a clone designated herein as“DNA26847-1395”.

FIG. 8 shows the amino acid sequence (SEQ ID NO:8) derived from thecoding sequence of SEQ ID NO:7 shown in FIG. 7.

FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a native sequencePRO182 cDNA, wherein SEQ ID NO:9 is a clone designated herein as“DNA27865-1091”.

FIG. 10 shows the amino acid sequence (SEQ ID NO:10) derived from thecoding sequence of SEQ ID NO:9 shown in FIG. 9.

FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a native sequencePRO205 cDNA, wherein SEQ ID NO:11 is a clone designated herein as“DNA30868-1156”.

FIG. 12 shows the amino acid sequence (SEQ ID NO:12) derived from thecoding sequence of SEQ ID NO:11 shown in FIG. 11.

FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a native sequencePRO204 cDNA, wherein SEQ ID NO:13 is a clone designated herein as“DNA30871-1157”.

FIG. 14 shows the amino acid sequence (SEQ ID NO:14) derived from thecoding sequence of SEQ ID NO:13 shown in FIG. 13.

FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a native sequencePRO1873 cDNA, wherein SEQ ID NO:15 is a clone designated herein as“DNA30880”.

FIG. 16 shows the amino acid sequence (SEQ ID NO:16) derived from thecoding sequence of SEQ ID NO:15 shown in FIG. 15.

FIG. 17 shows a nucleotide sequence (SEQ ID NO:17) of a native sequencePRO214 cDNA, wherein SEQ ID NO:17 is a clone designated herein as“DNA32286-1191”.

FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived from thecoding sequence of SEQ ID NO:17 shown in FIG. 17.

FIG. 19 shows a nucleotide sequence (SEQ ID NO:19) of a native sequencePRO221 cDNA, wherein SEQ ID NO:19 is a clone designated herein as“DNA33089-1132”.

FIG. 20 shows the amino acid sequence (SEQ ID NO:20) derived from thecoding sequence of SEQ ID NO:19 shown in FIG. 19.

FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a native sequencePRO228 cDNA, wherein SEQ ID NO:21 is a clone designated herein as“DNA33092-1202”.

FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived from thecoding sequence of SEQ ID NO:21 shown in FIG. 21.

FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a native sequencePRO229 cDNA, wherein SEQ ID NO:23 is a clone designated herein as“DNA33100-1159”.

FIG. 24 shows the amino acid sequence (SEQ ID NO:24) derived from thecoding sequence of SEQ ID NO:23 shown in FIG. 23.

FIG. 25 shows a nucleotide sequence (SEQ ID NO:25) of a native sequencePRO230 cDNA, wherein SEQ ID NO:25 is a clone designated herein as“DNA33223-1136”.

FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived from thecoding sequence of SEQ ID NO:25 shown in FIG. 25.

FIG. 27 shows a nucleotide sequence (SEQ ID NO:27) of a native sequencePRO7223 cDNA, wherein SEQ ID NO:27 is a clone designated herein as“DNA34385”.

FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived from thecoding sequence of SEQ ID NO:27 shown in FIG. 27.

FIG. 29 shows a nucleotide sequence (SEQ ID NO:29) of a native sequencePRO241 cDNA, wherein SEQ ID NO:29 is a clone designated herein as“DNA34392-1170”.

FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived from thecoding sequence of SEQ ID NO:29 shown in FIG. 29.

FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a native sequencePRO263 cDNA, wherein SEQ ID NO:31 is a clone designated herein as“DNA34431-1177”.

FIG. 32 shows the amino acid sequence (SEQ ID NO:32) derived from thecoding sequence of SEQ ID NO:31 shown in FIG. 31.

FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a native sequencePRO321 cDNA, wherein SEQ ID NO:33 is a clone designated herein as“DNA34433-1308”.

FIG. 34 shows the amino acid sequence (SEQ ID NO:34) derived from thecoding sequence of SEQ ID NO:33 shown in FIG. 33.

FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a native sequencePRO231 cDNA, wherein SEQ ID NO:35 is a clone designated herein as“DNA34434-1139”.

FIG. 36 shows the amino acid sequence (SEQ ID NO:36) derived from thecoding sequence of SEQ ID NO:35 shown in FIG. 35.

FIG. 37 shows a nucleotide sequence (SEQ ID NO:37) of a native sequencePRO238 cDNA, wherein SEQ ID NO:37 is a clone designated herein as“DNA35600-1162”.

FIG. 38 shows the amino acid sequence (SEQ ID NO:38) derived from thecoding sequence of SEQ ID NO:37 shown in FIG. 37.

FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a native sequencePRO247 cDNA, wherein SEQ ID NO:39 is a clone designated herein as“DNA35673-1201”.

FIG. 40 shows the amino acid sequence (SEQ ID NO:40) derived from thecoding sequence of SEQ ID NO:39 shown in FIG. 39.

FIG. 41 shows a nucleotide sequence (SEQ ID NO:41) of a native sequencePRO256 cDNA, wherein SEQ ID NO:41 is a clone designated herein as“DNA35880-1160”.

FIG. 42 shows the amino acid sequence (SEQ ID NO:42) derived from thecoding sequence of SEQ ID NO:41 shown in FIG. 41.

FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a native sequencePRO258 cDNA, wherein SEQ ID NO:43 is a clone designated herein as“DNA35918-1174”.

FIG. 44 shows the amino acid sequence (SEQ ID NO:44) derived from thecoding sequence of SEQ ID NO:43 shown in FIG. 43.

FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a native sequencePRO265 cDNA, wherein SEQ ID NO:45 is a clone designated herein as“DNA36350-1158”.

FIG. 46 shows the amino acid sequence (SEQ ID NO:46) derived from thecoding sequence of SEQ ID NO:45 shown in FIG. 45.

FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a native sequencePRO21 cDNA, wherein SEQ ID NO:47 is a clone designated herein as“DNA36638-1056”.

FIG. 48 shows the amino acid sequence (SEQ ID NO:48) derived from thecoding sequence of SEQ ID NO:47 shown in FIG. 47.

FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a native sequencePRO295 cDNA, wherein SEQ ID NO:49 is a clone designated herein as“DNA38268-1188”.

FIG. 50 shows the amino acid sequence (SEQ ID NO:50) derived from thecoding sequence of SEQ ID NO:49 shown in FIG. 49.

FIG. 51 shows a nucleotide sequence (SEQ ID NO:51) of a native sequencePRO302 cDNA, wherein SEQ ID NO:51 is a clone designated herein as“DNA40370-1217”.

FIG. 52 shows the amino acid sequence (SEQ ID NO:52) derived from thecoding sequence of SEQ ID NO:51 shown in FIG. 51.

FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a native sequencePRO301 cDNA, wherein SEQ ID NO:53 is a clone designated herein as“DNA40628-1216”.

FIG. 54 shows the amino acid sequence (SEQ ID NO:54) derived from thecoding sequence of SEQ ID NO:53 shown in FIG. 53.

FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a native sequencePRO337 cDNA, wherein SEQ ID NO:55 is a clone designated herein as“DNA43316-1237”.

FIG. 56 shows the amino acid sequence (SEQ ID NO:56) derived from thecoding sequence of SEQ ID NO:55 shown in FIG. 55.

FIG. 57 shows a nucleotide sequence (SEQ ID NO:57) of a native sequencePRO7248 cDNA, wherein SEQ ID NO:57 is a clone designated herein as“DNA44195”.

FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived from thecoding sequence of SEQ ID NO:57 shown in FIG. 57.

FIG. 59 shows a nucleotide sequence (SEQ ID NO:59) of a native sequencePRO846 cDNA, wherein SEQ ID NO:59 is a clone designated herein as“DNA44196-1353”.

FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived from thecoding sequence of SEQ ID NO:59 shown in FIG. 59.

FIG. 61 shows a nucleotide sequence (SEQ ID NO:61) of a native sequencePRO1864 cDNA, wherein SEQ ID NO:61 is a clone designated herein as“DNA45409-2511”.

FIG. 62 shows the amino acid sequence (SEQ ID NO:62) derived from thecoding sequence of SEQ ID NO:61 shown in FIG. 61.

FIG. 63 shows a nucleotide sequence (SEQ ID NO:63) of a native sequencePRO363 cDNA, wherein SEQ ID NO:63 is a clone designated herein as“DNA45419-1252”.

FIG. 64 shows the amino acid sequence (SEQ ID NO:64) derived from thecoding sequence of SEQ ID NO:63 shown in FIG. 63.

FIG. 65 shows a nucleotide sequence (SEQ ID NO:65) of a native sequencePRO730 cDNA, wherein SEQ ID NO:65 is a clone designated herein as“DNA45624-1400”.

FIG. 66 shows the amino acid sequence (SEQ ID NO:66) derived from thecoding sequence of SEQ ID NO:65 shown in FIG. 65.

FIG. 67 shows a nucleotide sequence (SEQ ID NO:67) of a native sequencePRO365 cDNA, wherein SEQ ID NO:67 is a clone designated herein as“DNA46777-1253”.

FIG. 68 shows the amino acid sequence (SEQ ID NO:68) derived from thecoding sequence of SEQ ID NO:67 shown in FIG. 67.

FIG. 69 shows a nucleotide sequence (SEQ ID NO:69) of a native sequencePRO532 cDNA, wherein SEQ ID NO:69 is a clone designated herein as“DNA48335”.

FIG. 70 shows the amino acid sequence (SEQ ID NO:70) derived from thecoding sequence of SEQ ID NO:69 shown in FIG. 69.

FIG. 71 shows a nucleotide sequence (SEQ ID NO:71) of a native sequencePRO322 cDNA, wherein SEQ ID NO:71 is a clone designated herein as“DNA48336-1309”.

FIG. 72 shows the amino acid sequence (SEQ ID NO:72) derived from thecoding sequence of SEQ ID NO:71 shown in FIG. 71.

FIG. 73 shows a nucleotide sequence (SEQ ID NO:73) of a native sequencePRO1120 cDNA, wherein SEQ ID NO:73 is a clone designated herein as“DNA48606-1479”.

FIG. 74 shows the amino acid sequence (SEQ ID NO:74) derived from thecoding sequence of SEQ ID NO:73 shown in FIG. 73.

FIG. 75 shows a nucleotide sequence (SEQ ID NO:75) of a native sequencePRO7261 cDNA, wherein SEQ ID NO:75 is a clone designated herein as“DNA49149”.

FIG. 76 shows the amino acid sequence (SEQ ID NO:76) derived from thecoding sequence of SEQ ID NO:75 shown in FIG. 75.

FIG. 77 shows a nucleotide sequence (SEQ ID NO:77) of a native sequencePRO533 cDNA, wherein SEQ ID NO:77 is a clone designated herein as“DNA49435-1219”.

FIG. 78 shows the amino acid sequence (SEQ ID NO:78) derived from thecoding sequence of SEQ ID NO:77 shown in FIG. 77.

FIG. 79 shows a nucleotide sequence (SEQ ID NO:79) of a native sequencePRO724 cDNA, wherein SEQ ID NO:79 is a clone designated herein as“DNA49631-1328”.

FIG. 80 shows the amino acid sequence (SEQ ID NO:80) derived from thecoding sequence of SEQ ID NO:79 shown in FIG. 79.

FIG. 81 shows a nucleotide sequence (SEQ ID NO:81) of a native sequencePRO734 cDNA, wherein SEQ ID NO:81 is a clone designated herein as“DNA49817”.

FIG. 82 shows the amino acid sequence (SEQ ID NO:82) derived from thecoding sequence of SEQ ID NO:81 shown in FIG. 81.

FIG. 83 shows a nucleotide sequence (SEQ ID NO:83) of a native sequencePRO771 cDNA, wherein SEQ ID NO:83 is a clone designated herein as“DNA49829-1346”.

FIG. 84 shows the amino acid sequence (SEQ ID NO:84) derived from thecoding sequence of SEQ ID NO:83 shown in FIG. 83.

FIG. 85 shows a nucleotide sequence (SEQ ID NO:85) of a native sequencePRO2010 cDNA, wherein SEQ ID NO:85 is a clone designated herein as“DNA50792”.

FIG. 86 shows the amino acid sequence (SEQ ID NO:86) derived from thecoding sequence of SEQ ID NO:85 shown in FIG. 85.

FIG. 87 shows a nucleotide sequence (SEQ ID NO:87) of a native sequencePRO871 cDNA, wherein SEQ ID NO:87 is a clone designated herein as“DNA50919-1361”.

FIG. 88 shows the amino acid sequence (SEQ ID NO:88) derived from thecoding sequence of SEQ ID NO:87 shown in FIG. 87.

FIG. 89 shows a nucleotide sequence (SEQ ID NO:89) of a native sequencePRO697 cDNA, wherein SEQ ID NO:89 is a clone designated herein as“DNA50920-1325”.

FIG. 90 shows the amino acid sequence (SEQ ID NO:90) derived from thecoding sequence of SEQ ID NO:89 shown in FIG. 89.

FIG. 91 shows a nucleotide sequence (SEQ ID NO:91) of a native sequencePRO1083 cDNA, wherein SEQ ID NO:91 is a clone designated herein as“DNA50921-1458”.

FIG. 92 shows the amino acid sequence (SEQ ID NO:22) derived from thecoding sequence of SEQ ID NO:91 shown in FIG. 91.

FIG. 93 shows a nucleotide sequence (SEQ ID NO:93) of a native sequencePRO725 cDNA, wherein SEQ ID NO:93 is a clone designated herein as“DNA52758-1399”.

FIG. 94 shows the amino acid sequence (SEQ ID NO:94) derived from thecoding sequence of SEQ ID NO:93 shown in FIG. 93.

FIG. 95 shows a nucleotide sequence (SEQ ID NO:95) of a native sequencePRO720 cDNA, wherein SEQ ID NO:95 is a clone designated herein as“DNA53517-1366-1”.

FIG. 96 shows the amino acid sequence (SEQ ID NO:96) derived from thecoding sequence of SEQ ID NO:95 shown in FIG. 95.

FIG. 97 shows a nucleotide sequence (SEQ ID NO:97) of a native sequencePRO738 cDNA, wherein SEQ ID NO:97 is a clone designated herein as“DNA53915-1258”.

FIG. 98 shows the amino acid sequence (SEQ ID NO:98) derived from thecoding sequence of SEQ ID NO:97 shown in FIG. 97.

FIG. 99 shows a nucleotide sequence (SEQ ID NO:99) of a native sequencePRO865 cDNA, wherein SEQ ID NO:99 is a clone designated herein as“DNA53974-1401”.

FIG. 100 shows the amino acid sequence (SEQ ID NO:100) derived from thecoding sequence of SEQ ID NO:99 shown in FIG. 99.

FIG. 101 shows a nucleotide sequence (SEQ ID NO:101) of a nativesequence PRO840 cDNA, wherein SEQ ID NO:101 is a clone designated hereinas “DNA53987-1438”.

FIG. 102 shows the amino acid sequence (SEQ ID NO:102) derived from thecoding sequence of SEQ ID NO:101 shown in FIG. 101.

FIG. 103 shows a nucleotide sequence (SEQ ID NO:103) of a nativesequence PRO1080 cDNA, wherein SEQ ID NO:103 is a clone designatedherein as “DNA56047-1456”.

FIG. 104 shows the amino acid sequence (SEQ ID NO:104) derived from thecoding sequence of SEQ ID NO:103 shown in FIG. 103.

FIG. 105 shows a nucleotide sequence (SEQ ID NO:105) of a nativesequence PRO1079 cDNA, wherein SEQ ID NO:105 is a clone designatedherein as “DNA56050-1455”.

FIG. 106 shows the amino acid sequence (SEQ ID NO:106) derived from thecoding sequence of SEQ ID NO:105 shown in FIG. 105.

FIG. 107 shows a nucleotide sequence (SEQ ID NO:107) of a nativesequence PRO793 cDNA, wherein SEQ ID NO:107 is a clone designated hereinas “DNA56110-1437”.

FIG. 108 shows the amino acid sequence (SEQ ID NO:108) derived from thecoding sequence of SEQ ID NO:107 shown in FIG. 107.

FIG. 109 shows a nucleotide sequence (SEQ ID NO:109) of a nativesequence PRO788 cDNA, wherein SEQ ID NO:109 is a clone designated hereinas “DNA56405-1357”.

FIG. 110 shows the amino acid sequence (SEQ ID NO:110) derived from thecoding sequence of SEQ ID NO:109 shown in FIG. 109.

FIG. 111 shows a nucleotide sequence (SEQ ID NO:111) of a nativesequence PRO938 cDNA, wherein SEQ ID NO:111 is a clone designated hereinas “DNA56433-1406”.

FIG. 112 shows the amino acid sequence (SEQ ID NO:112) derived from thecoding sequence of SEQ ID NO:111 shown in FIG. 111.

FIG. 113 shows a nucleotide sequence (SEQ ID NO:113) of a nativesequence PRO1012 cDNA, wherein SEQ ID NO:113 is a clone designatedherein as “DNA56439-1376”.

FIG. 114 shows the amino acid sequence (SEQ ID NO:114) derived from thecoding sequence of SEQ ID NO:113 shown in FIG. 113.

FIG. 115 shows a nucleotide sequence (SEQ ID NO:115) of a nativesequence PRO1477 cDNA, wherein SEQ ID NO:115 is a clone designatedherein as “DNA56529-1647”.

FIG. 116 shows the amino acid sequence (SEQ ID NO:116) derived from thecoding sequence of SEQ ID NO:115 shown in FIG. 115.

FIG. 117 shows a nucleotide sequence (SEQ ID NO:117) of a nativesequence PRO1134 cDNA, wherein SEQ ID NO:117 is a clone designatedherein as “DNA56865-1491”.

FIG. 118 shows the amino acid sequence (SEQ ID NO:118) derived from thecoding sequence of SEQ ID NO:117 shown in FIG. 117.

FIG. 119 shows a nucleotide sequence (SEQ ID NO:119) of a nativesequence PRO162 cDNA, wherein SEQ ID NO:119 is a clone designated hereinas “DNA56965-1356”.

FIG. 120 shows the amino acid sequence (SEQ ID NO:120) derived from thecoding sequence of SEQ ID NO:119 shown in FIG. 119.

FIG. 121 shows a nucleotide sequence (SEQ ID NO:121) of a nativesequence PRO1114 cDNA, wherein SEQ ID NO:121 is a clone designatedherein as “DNA57033-1403-1”.

FIG. 122 shows the amino acid sequence (SEQ ID NO:122) derived from thecoding sequence of SEQ ID NO:121 shown in FIG. 121.

FIG. 123 shows a nucleotide sequence (SEQ ID NO:123) of a nativesequence PRO828 cDNA, wherein SEQ ID NO:123 is a clone designated hereinas “DNA57037-1444”.

FIG. 124 shows the amino acid sequence (SEQ ID NO:124) derived from thecoding sequence of SEQ ID NO:123 shown in FIG. 123.

FIG. 125 shows a nucleotide sequence (SEQ ID NO:125) of a nativesequence PRO827 cDNA, wherein SEQ ID NO:125 is a clone designated hereinas “DNA57039-1402”.

FIG. 126 shows the amino acid sequence (SEQ ID NO:126) derived from thecoding sequence of SEQ ID NO:125 shown in FIG. 125.

FIG. 127 shows a nucleotide sequence (SEQ ID NO:127) of a nativesequence PRO1075 cDNA, wherein SEQ ID NO:127 is a clone designatedherein as “DNA57689-1385”.

FIG. 128 shows the amino acid sequence (SEQ ID NO:128) derived from thecoding sequence of SEQ ID NO:127 shown in FIG. 127.

FIG. 129 shows a nucleotide sequence (SEQ ID NO:129) of a nativesequence PRO1007 cDNA, wherein SEQ ID NO:129 is a clone designatedherein as “DNA57690-1374”.

FIG. 130 shows the amino acid sequence (SEQ ID NO:130) derived from thecoding sequence of SEQ ID NO:129 shown in FIG. 129.

FIG. 131 shows a nucleotide sequence (SEQ ID NO:131) of a nativesequence PRO826 cDNA, wherein SEQ ID NO:131 is a clone designated hereinas “DNA57694-1341”.

FIG. 132 shows the amino acid sequence (SEQ ID NO:132) derived from thecoding sequence of SEQ ID NO:131 shown in FIG. 131.

FIG. 133 shows a nucleotide sequence (SEQ ID NO:133) of a nativesequence PRO819 cDNA, wherein SEQ ID NO:132 is a clone designated hereinas “DNA57695-1340”.

FIG. 134 shows the amino acid sequence (SEQ ID NO:134) derived from thecoding sequence of SEQ ID NO:133 shown in FIG. 133.

FIG. 135 shows a nucleotide sequence (SEQ ID NO:135) of a nativesequence PRO1006 cDNA, wherein SEQ ID NO:135 is a clone designatedherein as “DNA57699-1412”.

FIG. 136 shows the amino acid sequence (SEQ ID NO:136) derived from thecoding sequence of SEQ ID NO:135 shown in FIG. 135.

FIG. 137 shows a nucleotide sequence (SEQ ID NO:137) of a nativesequence PRO982 cDNA, wherein SEQ ID NO:137 is a clone designated hereinas “DNA57700-1408”.

FIG. 138 shows the amino acid sequence (SEQ ID NO:138) derived from thecoding sequence of SEQ ID NO:137 shown in FIG. 137.

FIG. 139 shows a nucleotide sequence (SEQ ID NO:139) of a nativesequence PRO1005 cDNA, wherein SEQ ID NO:139 is a clone designatedherein as “DNA57708-1411”.

FIG. 140 shows the amino acid sequence (SEQ ID NO:140) derived from thecoding sequence of SEQ ID NO:139 shown in FIG. 139.

FIG. 141 shows a nucleotide sequence (SEQ ID NO:141) of a nativesequence PRO791 cDNA, wherein SEQ ID NO:141 is a clone designated hereinas “DNA57838-1337”.

FIG. 142 shows the amino acid sequence (SEQ ID NO:142) derived from thecoding sequence of SEQ ID NO:141 shown in FIG. 141.

FIG. 143 shows a nucleotide sequence (SEQ ID NO:143) of a nativesequence PRO1071 cDNA, wherein SEQ ID NO:143 is a clone designatedherein as “DNA58847-1383”.

FIG. 144 shows the amino acid sequence (SEQ ID NO:144) derived from thecoding sequence of SEQ ID NO:143 shown in FIG. 43.

FIG. 145 shows a nucleotide sequence (SEQ ID NO:145) of a nativesequence PRO1415 cDNA, wherein SEQ ID NO:145 is a clone designatedherein as “DNA58852-1637”.

FIG. 146 shows the amino acid sequence (SEQ ID NO:146) derived from thecoding sequence of SEQ ID NO:145 shown in FIG. 145.

FIG. 147 shows a nucleotide sequence (SEQ ID NO:147) of a nativesequence PRO1054 cDNA, wherein SEQ ID NO:147 is a clone designatedherein as “DNA58853-1423”.

FIG. 148 shows the amino acid sequence (SEQ ID NO:148) derived from thecoding sequence of SEQ ID NO:147 shown in FIG. 147.

FIG. 149 shows a nucleotide sequence (SEQ ID NO:149) of a nativesequence PRO1411 cDNA, wherein SEQ ID NO:149 is a clone designatedherein as “DNA59212-1627”.

FIG. 150 shows the amino acid sequence (SEQ ID NO:150) derived from thecoding sequence of SEQ ID NO:149 shown in FIG. 149.

FIG. 151 shows a nucleotide sequence (SEQ ID NO:151) of a nativesequence PRO1184 cDNA, wherein SEQ ID NO:151 is a clone designatedherein as “DNA59220-1514”.

FIG. 152 shows the amino acid sequence (SEQ ID NO:152) derived from thecoding sequence of SEQ ID NO:151 shown in FIG. 151.

FIG. 153 shows a nucleotide sequence (SEQ ID NO:153) of a nativesequence PRO1029 cDNA, wherein SEQ ID NO:153 is a clone designatedherein as “DNA59493-1420”.

FIG. 154 shows the amino acid sequence (SEQ ID NO:154) derived from thecoding sequence of SEQ ID NO:153 shown in FIG. 153.

FIG. 155 shows a nucleotide sequence (SEQ ID NO:155) of a nativesequence PRO1139 cDNA, wherein SEQ ID NO:155 is a clone designatedherein as “DNA59497-1496”.

FIG. 156 shows the amino acid sequence (SEQ ID NO:156) derived from thecoding sequence of SEQ ID NO:155 shown in FIG. 155.

FIG. 157 shows a nucleotide sequence (SEQ ID NO:157) of a nativesequence PRO1190 cDNA, wherein SEQ ID NO:157 is a clone designatedherein as “DNA59586-1520”.

FIG. 158 shows the amino acid sequence (SEQ ID NO:158) derived from thecoding sequence of SEQ ID NO:157 shown in FIG. 157.

FIG. 159 shows a nucleotide sequence (SEQ ID NO:159) of a nativesequence PRO1309 cDNA, wherein SEQ ID NO:159 is a clone designatedherein as “DNA59588-1571”.

FIG. 160 shows the amino acid sequence (SEQ ID NO:160) derived from thecoding sequence of SEQ ID NO:159 shown in FIG. 159.

FIG. 161 shows a nucleotide sequence (SEQ ID NO:161) of a nativesequence PRO836 cDNA, wherein SEQ ID NO:161 is a clone designated hereinas “DNA59620-1463”.

FIG. 162 shows the amino acid sequence (SEQ ID NO:162) derived from thecoding sequence of SEQ ID NO:161 shown in FIG. 161.

FIG. 163 shows a nucleotide sequence (SEQ ID NO:163) of a nativesequence PRO1025 cDNA, wherein SEQ ID NO:163 is a clone designatedherein as “DNA59622-1334”.

FIG. 164 shows the amino acid sequence (SEQ ID NO:164) derived from thecoding sequence of SEQ ID NO:163 shown in FIG. 163.

FIG. 165 shows a nucleotide sequence (SEQ ID NO:165) of a nativesequence PRO1131 cDNA, wherein SEQ ID NO:165 is a clone designatedherein as “DNA59777-1480”.

FIG. 166 shows the amino acid sequence (SEQ ID NO:166) derived from thecoding sequence of SEQ ID NO:165 shown in FIG. 165.

FIG. 167 shows a nucleotide sequence (SEQ ID NO:167) of a nativesequence PRO1182 cDNA, wherein SEQ ID NO:167 is a clone designatedherein as “DNA59848-1512”.

FIG. 168 shows the amino acid sequence (SEQ ID NO:168) derived from thecoding sequence of SEQ ID NO:167 shown in FIG. 167.

FIG. 169 shows a nucleotide sequence (SEQ ID NO:169) of a nativesequence PRO1155 cDNA, wherein SEQ ID NO:169 is a clone designatedherein as “DNA59849-1504”.

FIG. 170 shows the amino acid sequence (SEQ ID NO:170) derived from thecoding sequence of SEQ ID NO:169 shown in FIG. 169.

FIG. 171 shows a nucleotide sequence (SEQ ID NO:171) of a nativesequence PRO1186 cDNA, wherein SEQ ID NO:171 is a clone designatedherein as “DNA60621-1516”.

FIG. 172 shows the amino acid sequence (SEQ ID NO:172) derived from thecoding sequence of SEQ ID NO:171 shown in FIG. 171.

FIG. 173 shows a nucleotide sequence (SEQ ID NO:173) of a nativesequence PRO1198 cDNA, wherein SEQ ID NO:173 is a clone designatedherein as “DNA60622-1525”.

FIG. 174 shows the amino acid sequence (SEQ ID NO:174) derived from thecoding sequence of SEQ ID NO:173 shown in FIG. 173.

FIG. 175 shows a nucleotide sequence (SEQ ID NO:175) of a nativesequence PRO1265 cDNA, wherein SEQ ID NO:175 is a clone designatedherein as “DNA60764-1533”.

FIG. 176 shows the amino acid sequence (SEQ ID NO:176) derived from thecoding sequence of SEQ ID NO:175 shown in FIG. 175.

FIG. 177 shows a nucleotide sequence (SEQ ID NO:177) of a nativesequence PRO1361 cDNA, wherein SEQ ID NO:177 is a clone designatedherein as “DNA60783-1611”.

FIG. 178 shows the amino acid sequence (SEQ ID NO:178) derived from thecoding sequence of SEQ ID NO:177 shown in FIG. 177.

FIG. 179 shows a nucleotide sequence (SEQ ID NO:179) of a nativesequence PRO1287 cDNA, wherein SEQ ID NO:179 is a clone designatedherein as “DNA61755-1554”.

FIG. 180 shows the amino acid sequence (SEQ ID NO:180) derived from thecoding sequence of SEQ ID NO:179 shown in FIG. 179.

FIG. 181 shows a nucleotide sequence (SEQ ID NO:181) of a nativesequence PRO1308 cDNA, wherein SEQ ID NO:181 is a clone designatedherein as “DNA62306-1570”.

FIG. 182 shows the amino acid sequence (SEQ ID NO:182) derived from thecoding sequence of SEQ ID NO:181 shown in FIG. 181.

FIG. 183 shows a nucleotide sequence (SEQ ID NO:183) of a nativesequence PRO4313 cDNA, wherein SEQ ID NO:183 is a clone designatedherein as “DNA62312-2558”.

FIG. 184 shows the amino acid sequence (SEQ ID NO:184) derived from thecoding sequence of SEQ ID NO:183 shown in FIG. 183.

FIG. 185 shows a nucleotide sequence (SEQ ID NO:185) of a nativesequence PRO1192 cDNA, wherein SEQ ID NO:185 is a clone designatedherein as “DNA62814-1521”.

FIG. 186 shows the amino acid sequence (SEQ ID NO:186) derived from thecoding sequence of SEQ ID NO:185 shown in FIG. 185.

FIG. 187 shows a nucleotide sequence (SEQ ID NO:187) of a nativesequence PRO1160 cDNA, wherein SEQ ID NO:187 is a clone designatedherein as “DNA62872-1509”.

FIG. 188 shows the amino acid sequence (SEQ ID NO:188) derived from thecoding sequence of SEQ ID NO:187 shown in FIG. 187.

FIG. 189 shows a nucleotide sequence (SEQ ID NO:189) of a nativesequence PRO1244 cDNA, wherein SEQ ID NO:189 is a clone designatedherein as “DNA64883-1526”.

FIG. 190 shows the amino acid sequence (SEQ ID NO:190) derived from thecoding sequence of SEQ ID NO:189 shown in FIG. 189.

FIG. 191 shows a nucleotide sequence (SEQ ID NO:191) of a nativesequence PRO1356 cDNA, wherein SEQ ID NO:191 is a clone designatedherein as “DNA64886-1601”.

FIG. 192 shows the amino acid sequence (SEQ ID NO:192) derived from thecoding sequence of SEQ ID NO:191 shown in FIG. 191.

FIG. 193 shows a nucleotide sequence (SEQ ID NO:193) of a nativesequence PRO1274 cDNA, wherein SEQ ID NO:193 is a clone designatedherein as “DNA64889-1541”.

FIG. 194 shows the amino acid sequence (SEQ ID NO:194) derived from thecoding sequence of SEQ ID NO:193 shown in FIG. 193.

FIG. 195 shows a nucleotide sequence (SEQ ID NO:195) of a nativesequence PRO1272 cDNA, wherein SEQ ID NO:195 is a clone designatedherein as “DNA64896-1539”.

FIG. 196 shows the amino acid sequence (SEQ ID NO:196) derived from thecoding sequence of SEQ ID NO:195 shown in FIG. 195.

FIG. 197 shows a nucleotide sequence (SEQ ID NO:197) of a nativesequence PRO1412 cDNA, wherein SEQ ID NO:197 is a clone designatedherein as “DNA64897-1628”.

FIG. 198 shows the amino acid sequence (SEQ ID NO:198) derived from thecoding sequence of SEQ ID NO:197 shown in FIG. 197.

FIG. 199 shows a nucleotide sequence (SEQ ID NO:199) of a nativesequence PRO1286 cDNA, wherein SEQ ID NO:199 is a clone designatedherein as “DNA64903-1553”.

FIG. 200 shows the amino acid sequence (SEQ ID NO:200) derived from thecoding sequence of SEQ ID NO:199 shown in FIG. 199.

FIG. 201 shows a nucleotide sequence (SEQ ID NO:201) of a nativesequence PRO1347 cDNA, wherein SEQ ID NO:201 is a clone designatedherein as “DNA64950-1590”.

FIG. 202 shows the amino acid sequence (SEQ ID NO:202) derived from thecoding sequence of SEQ ID NO:201 shown in FIG. 201.

FIG. 203 shows a nucleotide sequence (SEQ ID NO:203) of a nativesequence PRO1273 cDNA, wherein SEQ ID NO:203 is a clone designatedherein as “DNA65402-1540”.

FIG. 204 shows the amino acid sequence (SEQ ID NO:204) derived from thecoding sequence of SEQ ID NO:203 shown in FIG. 203.

FIG. 205 shows a nucleotide sequence (SEQ ID NO:205) of a nativesequence PRO1283 cDNA, wherein SEQ ID NO:205 is a clone designatedherein as “DNA65404-1551”.

FIG. 206 shows the amino acid sequence (SEQ ID NO:206) derived from thecoding sequence of SEQ ID NO:205 shown in FIG. 205.

FIG. 207 shows a nucleotide sequence (SEQ ID NO:207) of a nativesequence PRO1279 cDNA, wherein SEQ ID NO:207 is a clone designatedherein as “DNA65405-1547”.

FIG. 208 shows the amino acid sequence (SEQ ID NO:208) derived from thecoding sequence of SEQ ID NO:207 shown in FIG. 207.

FIG. 209 shows a nucleotide sequence (SEQ ID NO:209) of a nativesequence PRO1306 cDNA, wherein SEQ ID NO:209 is a clone designatedherein as “DNA65410-1569”.

FIG. 210 shows the amino acid sequence (SEQ ID NO:210) derived from thecoding sequence of SEQ ID NO:209 shown in FIG. 209.

FIG. 211 shows a nucleotide sequence (SEQ ID NO:211) of a nativesequence PRO1195 cDNA, wherein SEQ ID NO:211 is a clone designatedherein as “DNA65412-1523”.

FIG. 212 shows the amino acid sequence (SEQ ID NO:212) derived from thecoding sequence of SEQ ID NO:211 shown in FIG. 211.

FIG. 213 shows a nucleotide sequence (SEQ ID NO:213) of a nativesequence PRO4995 cDNA, wherein SEQ ID NO:213 is a clone designatedherein as “DNA66307-2661”.

FIG. 214 shows the amino acid sequence (SEQ ID NO:214) derived from thecoding sequence of SEQ ID NO:213 shown in FIG. 213.

FIG. 215 shows a nucleotide sequence (SEQ ID NO:215) of a nativesequence PRO1382 cDNA, wherein SEQ ID NO:215 is a clone designatedherein as “DNA66526-1616”.

FIG. 216 shows the amino acid sequence (SEQ ID NO:216) derived from thecoding sequence of SEQ ID NO:215 shown in FIG. 215.

FIG. 217 shows a nucleotide sequence (SEQ ID NO:217) of a nativesequence PRO1325 cDNA, wherein SEQ ID NO:217 is a clone designatedherein as “DNA66659-1593”.

FIG. 218 shows the amino acid sequence (SEQ ID NO:218) derived from thecoding sequence of SEQ ID NO:217 shown in FIG. 217.

FIG. 219 shows a nucleotide sequence (SEQ ID NO:219) of a nativesequence PRO1329 cDNA, wherein SEQ ID NO:219 is a clone designatedherein as “DNA66660-1585”.

FIG. 220 shows the amino acid sequence (SEQ ID NO:220) derived from thecoding sequence of SEQ ID NO:219 shown in FIG. 219.

FIG. 221 shows a nucleotide sequence (SEQ ID NO:221) of a nativesequence PRO1338 cDNA, wherein SEQ ID NO:221 is a clone designatedherein as “DNA66667-1596”.

FIG. 222 shows the amino acid sequence (SEQ ID NO:222) derived from thecoding sequence of SEQ ID NO:221 shown in FIG. 221.

FIG. 223 shows a nucleotide sequence (SEQ ID NO:223) of a nativesequence PRO1337 cDNA, wherein SEQ ID NO:223 is a clone designatedherein as “DNA66672-1586”.

FIG. 224 shows the amino acid sequence (SEQ ID NO:224) derived from thecoding sequence of SEQ ID NO:223 shown in FIG. 223.

FIG. 225 shows a nucleotide sequence (SEQ ID NO:225) of a nativesequence PRO1343 cDNA, wherein SEQ ID NO:225 is a clone designatedherein as “DNA66675-1587”.

FIG. 226 shows the amino acid sequence (SEQ ID NO:226) derived from thecoding sequence of SEQ ID NO:225 shown in FIG. 225.

FIG. 227 shows a nucleotide sequence (SEQ ID NO:227) of a nativesequence PRO1376 cDNA, wherein SEQ ID NO:227 is a clone designatedherein as “DNA67300-1605”.

FIG. 228 shows the amino acid sequence (SEQ ID NO:228) derived from thecoding sequence of SEQ ID NO:227 shown in FIG. 227.

FIG. 229 shows a nucleotide sequence (SEQ ID NO:229) of a nativesequence PRO1434 cDNA, wherein SEQ ID NO:229 is a clone designatedherein as “DNA68818-2536”.

FIG. 230 shows the amino acid sequence (SEQ ID NO:230) derived from thecoding sequence of SEQ ID NO:229 shown in FIG. 229.

FIG. 231 shows a nucleotide sequence (SEQ ID NO:231) of a nativesequence PRO3579 cDNA, wherein SEQ ID NO:231 is a clone designatedherein as “DNA68862-2546”.

FIG. 232 shows the amino acid sequence (SEQ ID NO:232) derived from thecoding sequence of SEQ ID NO:231 shown in FIG. 231.

FIG. 233 shows a nucleotide sequence (SEQ ID NO:233) of a nativesequence PRO1387 cDNA, wherein SEQ ID NO:233 is a clone designatedherein as “DNA68872-1620”.

FIG. 234 shows the amino acid sequence (SEQ ID NO:234) derived from thecoding sequence of SEQ ID NO:233 shown in FIG. 233.

FIG. 235 shows a nucleotide sequence (SEQ ID NO:235) of a nativesequence PRO1419 cDNA, wherein SEQ ID NO:235 is a clone designatedherein as “DNA71290-1630”.

FIG. 236 shows the amino acid sequence (SEQ ID NO:236) derived from thecoding sequence of SEQ ID NO:235 shown in FIG. 235.

FIG. 237 shows a nucleotide sequence (SEQ ID NO:237) of a nativesequence PRO1488 cDNA, wherein SEQ ID NO:237 is a clone designatedherein as “DNA73736-1657”.

FIG. 238 shows the amino acid sequence (SEQ ID NO:238) derived from thecoding sequence of SEQ ID NO:237 shown in FIG. 237.

FIG. 239 shows a nucleotide sequence (SEQ ID NO:239) of a nativesequence PRO1474 cDNA, wherein SEQ ID NO:239 is a clone designatedherein as “DNA73739-1645”.

FIG. 240 shows the amino acid sequence (SEQ ID NO:240) derived from thecoding sequence of SEQ ID NO:239 shown in FIG. 239.

FIG. 241 shows a nucleotide sequence (SEQ ID NO:241) of a nativesequence PRO1508 cDNA, wherein SEQ ID NO:241 is a clone designatedherein as “DNA73742-1662”.

FIG. 242 shows the amino acid sequence (SEQ ID NO:242) derived from thecoding sequence of SEQ ID NO:241 shown in FIG. 241.

FIG. 243 shows a nucleotide sequence (SEQ ID NO:243) of a nativesequence PRO1754 cDNA, wherein SEQ ID NO:243 is a clone designatedherein as “DNA76385-1692”.

FIG. 244 shows the amino acid sequence (SEQ ID NO:244) derived from thecoding sequence of SEQ ID NO:243 shown in FIG. 243.

FIG. 245 shows a nucleotide sequence (SEQ ID NO:245) of a nativesequence PRO1550 cDNA, wherein SEQ ID NO:245 is a clone designatedherein as “DNA76393-1664”.

FIG. 246 shows the amino acid sequence (SEQ ID NO:246) derived from thecoding sequence of SEQ ID NO:245 shown in FIG. 245.

FIG. 247 shows a nucleotide sequence (SEQ ID NO:247) of a nativesequence PRO1758 cDNA, wherein SEQ ID NO:247 is a clone designatedherein as “DNA76399-1700”.

FIG. 248 shows the amino acid sequence (SEQ ID NO:248) derived from thecoding sequence of SEQ ID NO:247 shown in FIG. 247.

FIG. 249 shows a nucleotide sequence (SEQ ID NO:249) of a nativesequence PRO1917 cDNA, wherein SEQ ID NO:249 is a clone designatedherein as “DNA76400-2528”.

FIG. 250 shows the amino acid sequence (SEQ ID NO:250) derived from thecoding sequence of SEQ ID NO:249 shown in FIG. 249.

FIG. 251 shows a nucleotide sequence (SEQ ID NO:251) of a nativesequence PRO1787 cDNA, wherein SEQ ID NO:251 is a clone designatedherein as “DNA76510-2504”.

FIG. 252 shows the amino acid sequence (SEQ ID NO:252) derived from thecoding sequence of SEQ ID NO:251 shown in FIG. 251.

FIG. 253 shows a nucleotide sequence (SEQ ID NO:253) of a nativesequence PRO1556 cDNA, wherein SEQ ID NO:253 is a clone designatedherein as “DNA76529-1666”.

FIG. 254 shows the amino acid sequence (SEQ ID NO:254) derived from thecoding sequence of SEQ ID NO:253 shown in FIG. 253.

FIG. 255 shows a nucleotide sequence (SEQ ID NO:255) of a nativesequence PRO1760 cDNA, wherein SEQ ID NO:255 is a clone designatedherein as “DNA76532-1702”.

FIG. 256 shows the amino acid sequence (SEQ ID NO:256) derived from thecoding sequence of SEQ ID NO:255 shown in FIG. 255.

FIG. 257 shows a nucleotide sequence (SEQ ID NO:257) of a nativesequence PRO1567 cDNA, wherein SEQ ID NO:257 is a clone designatedherein as “DNA76541-1675”.

FIG. 258 shows the amino acid sequence (SEQ ID NO:258) derived from thecoding sequence of SEQ ID NO:257 shown in FIG. 257.

FIG. 259 shows a nucleotide sequence (SEQ ID NO:259) of a nativesequence PRO1600 cDNA, wherein SEQ ID NO:259 is a clone designatedherein as “DNA77503-1686”.

FIG. 260 shows the amino acid sequence (SEQ ID NO:260) derived from thecoding sequence of SEQ ID NO:259 shown in FIG. 259.

FIG. 261 shows a nucleotide sequence (SEQ ID NO:261) of a nativesequence PRO1868 cDNA, wherein SEQ ID NO:261 is a clone designatedherein as “DNA77624-2515”.

FIG. 262 shows the amino acid sequence (SEQ ID NO:262) derived from thecoding sequence of SEQ ID NO:261 shown in FIG. 261.

FIG. 263 shows a nucleotide sequence (SEQ ID NO:263) of a nativesequence PRO1890 cDNA, wherein SEQ ID NO:263 is a clone designatedherein as “DNA79230-2525”.

FIG. 264 shows the amino acid sequence (SEQ ID NO:264) derived from thecoding sequence of SEQ ID NO:263 shown in FIG. 263.

FIG. 265 shows a nucleotide sequence (SEQ ID NO:265) of a nativesequence PRO1887 cDNA, wherein SEQ ID NO:265 is a clone designatedherein as “DNA79862-2522”.

FIG. 266 shows the amino acid sequence (SEQ ID NO:265) derived from thecoding sequence of SEQ ID NO:265 shown in FIG. 265.

FIG. 267 shows a nucleotide sequence (SEQ ID NO:267) of a nativesequence PRO4353 cDNA, wherein SEQ ID NO:267 is a clone designatedherein as “DNA80145-2594”.

FIG. 268 shows the amino acid sequence (SEQ ID NO:268) derived from thecoding sequence of SEQ ID NO:267 shown in FIG. 267.

FIG. 269 shows a nucleotide sequence (SEQ ID NO:269) of a nativesequence PRO1782 cDNA, wherein SEQ ID NO:269 is a clone designatedherein as “DNA80899-2501”.

FIG. 270 shows the amino acid sequence (SEQ ID NO:270) derived from thecoding sequence of SEQ ID NO:269 shown in FIG. 269.

FIG. 271 shows a nucleotide sequence (SEQ ID NO:271) of a nativesequence PRO1928 cDNA, wherein SEQ ID NO:271 is a clone designatedherein as “DNA81754-2532”.

FIG. 272 shows the amino acid sequence (SEQ ID NO:272) derived from thecoding sequence of SEQ ID NO:271 shown in FIG. 271.

FIG. 273 shows a nucleotide sequence (SEQ ID NO:273) of a nativesequence PRO1865 cDNA, wherein SEQ ID NO:273 is a clone designatedherein as “DNA81757-2512”.

FIG. 274 shows the amino acid sequence (SEQ ID NO:274) derived from thecoding sequence of SEQ ID NO:273 shown in FIG. 273.

FIG. 275 shows a nucleotide sequence (SEQ ID NO:275) of a nativesequence PRO4341 cDNA, wherein SEQ ID NO:275 is a clone designatedherein as “DNA81761-2583”.

FIG. 276 shows the amino acid sequence (SEQ ID NO:276) derived from thecoding sequence of SEQ ID NO:275 shown in FIG. 275.

FIG. 277 shows a nucleotide sequence (SEQ ID NO:277) of a nativesequence PRO6714 cDNA, wherein SEQ ID NO:277 is a clone designatedherein as “DNA82358-2738”.

FIG. 278 shows the amino acid sequence (SEQ ID NO:278) derived from thecoding sequence of SEQ ID NO:277 shown in FIG. 277.

FIG. 279 shows a nucleotide sequence (SEQ ID NO:279) of a nativesequence PRO5723 cDNA, wherein SEQ ID NO:279 is a clone designatedherein as “DNA82361”.

FIG. 280 shows the amino acid sequence (SEQ ID NO:280) derived from thecoding sequence of SEQ ID NO:279 shown in FIG. 279.

FIG. 281 shows a nucleotide sequence (SEQ ID NO:281) of a nativesequence PRO3438 cDNA, wherein SEQ ID NO:281 is a clone designatedherein as “DNA82364-2538”.

FIG. 282 shows the amino acid sequence (SEQ ID NO:282) derived from thecoding sequence of SEQ ID NO:281 shown in FIG. 281.

FIG. 283 shows a nucleotide sequence (SEQ ID NO:283) of a nativesequence PRO6071 cDNA, wherein SEQ ID NO:283 is a clone designatedherein as “DNA82403-2959”.

FIG. 284 shows the amino acid sequence (SEQ ID NO:284) derived from thecoding sequence of SEQ ID NO:283 shown in FIG. 283.

FIG. 285 shows a nucleotide sequence (SEQ ID NO:285) of a nativesequence PRO1801 cDNA, wherein SEQ ID NO:285 is a clone designatedherein as “DNA83500-2506”.

FIG. 286 shows the amino acid sequence (SEQ ID NO:286) derived from thecoding sequence of SEQ ID NO:285 shown in FIG. 285.

FIG. 287 shows a nucleotide sequence (SEQ ID NO:287) of a nativesequence PRO4324 cDNA, wherein SEQ ID NO:287 is a clone designatedherein as “DNA83560-2569”.

FIG. 288 shows the amino acid sequence (SEQ ID NO:288) derived from thecoding sequence of SEQ ID NO:287 shown in FIG. 287.

FIG. 289 shows a nucleotide sequence (SEQ ID NO:289) of a nativesequence PRO4333 cDNA, wherein SEQ ID NO:289 is a clone designatedherein as “DNA84210-2576”.

FIG. 290 shows the amino acid sequence (SEQ ID NO:290) derived from thecoding sequence of SEQ ID NO:289 shown in FIG. 289.

FIG. 291 shows a nucleotide sequence (SEQ ID NO:291) of a nativesequence PRO4405 cDNA, wherein SEQ ID NO:291 is a clone designatedherein as “DNA84920-2614”.

FIG. 292 shows the amino acid sequence (SEQ ID NO:292) derived from thecoding sequence of SEQ ID NO:291 shown in FIG. 291.

FIG. 293 shows a nucleotide sequence (SEQ ID NO:293) of a nativesequence PRO4356 cDNA, wherein SEQ ID NO:293 is a clone designatedherein as “DNA86576-2595”.

FIG. 294 shows the amino acid sequence (SEQ ID NO:294) derived from thecoding sequence of SEQ ID NO:293 shown in FIG. 293.

FIG. 295 shows a nucleotide sequence (SEQ ID NO:295) of a nativesequence PRO3444 cDNA, wherein SEQ ID NO:295 is a clone designatedherein as “DNA87997”.

FIG. 296 shows the amino acid sequence (SEQ ID NO:296) derived from thecoding sequence of SEQ ID NO:295 shown in FIG. 295.

FIG. 297 shows a nucleotide sequence (SEQ ID NO:297) of a nativesequence PRO4302 cDNA, wherein SEQ ID NO:297 is a clone designatedherein as “DNA92218-2554”.

FIG. 298 shows the amino acid sequence (SEQ ID NO:298) derived from thecoding sequence of SEQ ID NO:297 shown in FIG. 297.

FIG. 299 shows a nucleotide sequence (SEQ ID NO:299) of a nativesequence PRO4371 cDNA, wherein SEQ ID NO:299 is a clone designatedherein as “DNA92233-2599”.

FIG. 300 shows the amino acid sequence (SEQ ID NO:300) derived from thecoding sequence of SEQ ID NO:299 shown in FIG. 299.

FIG. 301 shows a nucleotide sequence (SEQ ID NO:301) of a nativesequence PRO4354 cDNA, wherein SEQ ID NO:301 is a clone designatedherein as “DNA92256-2596”.

FIG. 302 shows the amino acid sequence (SEQ ID NO:302) derived from thecoding sequence of SEQ ID NO:301 shown in FIG. 301.

FIG. 303 shows a nucleotide sequence (SEQ ID NO:303) of a nativesequence PRO5725 cDNA, wherein SEQ ID NO:303 is a clone designatedherein as “DNA92265-2669”.

FIG. 304 shows the amino acid sequence (SEQ ID NO:304) derived from thecoding sequence of SEQ ID NO:303 shown in FIG. 303.

FIG. 305 shows a nucleotide sequence (SEQ ID NO:305) of a nativesequence PRO4408 cDNA, wherein SEQ ID NO:305 is a clone designatedherein as “DNA92274-2617”.

FIG. 306 shows the amino acid sequence (SEQ ID NO:306) derived from thecoding sequence of SEQ ID NO:305 shown in FIG. 305.

FIG. 307 shows a nucleotide sequence (SEQ ID NO:307) of a nativesequence PRO9940 cDNA, wherein SEQ ID NO:307 is a clone designatedherein as “DNA92282”.

FIG. 308 shows the amino acid sequence (SEQ ID NO:308) derived from thecoding sequence of SEQ ID NO:307 shown in FIG. 307.

FIG. 309 shows a nucleotide sequence (SEQ ID NO:309) of a nativesequence PRO5737 cDNA, wherein SEQ ID NO:309 is a clone designatedherein as “DNA92929-2534-1”.

FIG. 310 shows the amino acid sequence (SEQ ID NO:310) derived from thecoding sequence of SEQ ID NO:309 shown in FIG. 309.

FIG. 311 shows a nucleotide sequence (SEQ ID NO:311) of a nativesequence PRO4425 cDNA, wherein SEQ ID NO:311 is a clone designatedherein as “DNA93011-2637”.

FIG. 312 shows the amino acid sequence (SEQ ID NO:312) derived from thecoding sequence of SEQ ID NO:311 shown in FIG. 311.

FIG. 313 shows a nucleotide sequence (SEQ ID NO:313) of a nativesequence PRO4345 cDNA, wherein SEQ ID NO:313 is a clone designatedherein as “DNA94854-2586”.

FIG. 314 shows the amino acid sequence (SEQ ID NO:314) derived from thecoding sequence of SEQ ID NO:313 shown in FIG. 313.

FIG. 315 shows a nucleotide sequence (SEQ ID NO:315) of a nativesequence PRO4342 cDNA, wherein SEQ ID NO:315 is a clone designatedherein as “DNA96787-2534-1”.

FIG. 316 shows the amino acid sequence (SEQ ID NO:316) derived from thecoding sequence of SEQ ID NO:315 shown in FIG. 315.

FIG. 317 shows a nucleotide sequence (SEQ ID NO:317) of a nativesequence PRO3562 cDNA, wherein SEQ ID NO:317 is a clone designatedherein as “DNA96791”.

FIG. 318 shows the amino acid sequence (SEQ ID NO:318) derived from thecoding sequence of SEQ ID NO:317 shown in FIG. 317.

FIG. 319 shows a nucleotide sequence (SEQ ID NO:319) of a nativesequence PRO4422 cDNA, wherein SEQ ID NO:319 is a clone designatedherein as “DNA96867-2620”.

FIG. 320 shows the amino acid sequence (SEQ ID NO:320) derived from thecoding sequence of SEQ ID NO:319 shown in FIG. 319.

FIG. 321 shows a nucleotide sequence (SEQ ID NO:321) of a nativesequence PRO5776 cDNA, wherein SEQ ID NO:321 is a clone designatedherein as “DNA96872-2674”.

FIG. 322 shows the amino acid sequence (SEQ ID NO:322) derived from thecoding sequence of SEQ ID NO:321 shown in FIG. 321.

FIG. 323 shows a nucleotide sequence (SEQ ID NO:323) of a nativesequence PRO4430 cDNA, wherein SEQ ID NO:323 is a clone designatedherein as “DNA96878-2626”.

FIG. 324 shows the amino acid sequence (SEQ ID NO:324) derived from thecoding sequence of SEQ ID NO:323 shown in FIG. 323.

FIG. 325 shows a nucleotide sequence (SEQ ID NO:325) of a nativesequence PRO4499 cDNA, wherein SEQ ID NO:325 is a clone designatedherein as “DNA96889-2641”.

FIG. 326 shows the amino acid sequence (SEQ ID NO:326) derived from thecoding sequence of SEQ ID NO:325 shown in FIG. 325.

FIG. 327 shows a nucleotide sequence (SEQ ID NO:327) of a nativesequence PRO4503 cDNA, wherein SEQ ID NO:327 is a clone designatedherein as “DNA100312-2645”.

FIG. 328 shows the amino acid sequence (SEQ ID NO:328) derived from thecoding sequence of SEQ ID NO:327 shown in FIG. 327.

FIG. 329 shows a nucleotide sequence (SEQ ID NO:329) of a nativesequence PRO10008 cDNA, wherein SEQ ID NO:329 is a clone designatedherein as “DNA101921”.

FIG. 330 shows the amino acid sequence (SEQ ID NO:330) derived from thecoding sequence of SEQ ID NO:329 shown in FIG. 329.

FIG. 331 shows a nucleotide sequence (SEQ ID NO:331) of a nativesequence PRO5730 cDNA, wherein SEQ ID NO:331 is a clone designatedherein as “DNA101926”.

FIG. 332 shows the amino acid sequence (SEQ ID NO:332) derived from thecoding sequence of SEQ ID NO:331 shown in FIG. 331.

FIG. 333 shows a nucleotide sequence (SEQ ID NO:333) of a nativesequence PRO6008 cDNA, wherein SEQ ID NO:333 is a clone designatedherein as “DNA102844”.

FIG. 334 shows the amino acid sequence (SEQ ID NO:334) derived from thecoding sequence of SEQ ID NO:333 shown in FIG. 333.

FIG. 335 shows a nucleotide sequence (SEQ ID NO:335) of a nativesequence PRO4527 cDNA, wherein SEQ ID NO:335 is a clone designatedherein as “DNA103197”.

FIG. 336 shows the amino acid sequence (SEQ ID NO:336) derived from thecoding sequence of SEQ ID NO:335 shown in FIG. 335.

FIG. 337 shows a nucleotide sequence (SEQ ID NO:337) of a nativesequence PRO4538 cDNA, wherein SEQ ID NO:337 is a clone designatedherein as “DNA103208”.

FIG. 338 shows the amino acid sequence (SEQ ID NO:338) derived from thecoding sequence of SEQ ID NO:337 shown in FIG. 337.

FIG. 339 shows a nucleotide sequence (SEQ ID NO:339) of a nativesequence PRO4553 cDNA, wherein SEQ ID NO:339 is a clone designatedherein as “DNA103223”.

FIG. 340 shows the amino acid sequence (SEQ ID NO:340) derived from thecoding sequence of SEQ ID NO:339 shown in FIG. 339.

FIG. 341 shows a nucleotide sequence (SEQ ID NO:341) of a nativesequence PRO6006 cDNA, wherein SEQ ID NO:341 is a clone designatedherein as “DNA 105782-2693”.

FIG. 342 shows the amino acid sequence (SEQ ID NO:342) derived from thecoding sequence of SEQ ID NO:341 shown in FIG. 341.

FIG. 343 shows a nucleotide sequence (SEQ ID NO:343) of a nativesequence PRO6029 cDNA, wherein SEQ ID NO:343 is a clone designatedherein as “DNA105849-2704”.

FIG. 344 shows the amino acid sequence (SEQ ID NO:344) derived from thecoding sequence of SEQ ID NO:343 shown in FIG. 343.

FIG. 345 shows a nucleotide sequence (SEQ ID NO:345) of a nativesequence PRO9821 cDNA, wherein SEQ ID NO:345 is a clone designatedherein as “DNA108725-2766”.

FIG. 346 shows the amino acid sequence (SEQ ID NO:346) derived from thecoding sequence of SEQ ID NO:345 shown in FIG. 345.

FIG. 347 shows a nucleotide sequence (SEQ ID NO:347) of a nativesequence PRO9820 cDNA, wherein SEQ ID NO:347 is a clone designatedherein as “DNA108769-2765”.

FIG. 348 shows the amino acid sequence (SEQ ID NO:348) derived from thecoding sequence of SEQ ID NO:347 shown in FIG. 347.

FIG. 349 shows a nucleotide sequence (SEQ ID NO:349) of a nativesequence PRO9771 cDNA, wherein SEQ ID NO:349 is a clone designatedherein as “DNA119498-2965”.

FIG. 350 shows the amino acid sequence (SEQ ID NO:350) derived from thecoding sequence of SEQ ID NO:349 shown in FIG. 349.

FIG. 351 shows a nucleotide sequence (SEQ ID NO:351) of a nativesequence PRO7436 cDNA, wherein SEQ ID NO:351 is a clone designatedherein as “DNA119535-2756”.

FIG. 352 shows the amino acid sequence (SEQ ID NO:352) derived from thecoding sequence of SEQ ID NO:351 shown in FIG. 351.

FIG. 353 shows a nucleotide sequence (SEQ ID NO:353) of a nativesequence PRO10096 cDNA, wherein SEQ ID NO:353 is a clone designatedherein as “DNA125185-2806”.

FIG. 354 shows the amino acid sequence (SEQ ID NO:354) derived from thecoding sequence of SEQ ID NO:353 shown in FIG. 353.

FIG. 355 shows a nucleotide sequence (SEQ ID NO:355) of a nativesequence PRO19670 cDNA, wherein SEQ ID NO:355 is a clone designatedherein as “DNA131639-2874”.

FIG. 356 shows the amino acid sequence (SEQ ID NO:356) derived from thecoding sequence of SEQ ID NO:355 shown in FIG. 355.

FIG. 357 shows a nucleotide sequence (SEQ ID NO:357) of a nativesequence PRO20044 cDNA, wherein SEQ ID NO:357 is a clone designatedherein as “DNA139623-2893”.

FIG. 358 shows the amino acid sequence (SEQ ID NO:358) derived from thecoding sequence of SEQ ID NO:357 shown in FIG. 357.

FIG. 359 shows a nucleotide sequence (SEQ ID NO:359) of a nativesequence PRO9873 cDNA, wherein SEQ ID NO:359 is a clone designatedherein as “DNA143076-2787”.

FIG. 360 shows the amino acid sequence (SEQ ID NO:360) derived from thecoding sequence of SEQ ID NO:359 shown in FIG. 359.

FIG. 361 shows a nucleotide sequence (SEQ ID NO:361) of a nativesequence PRO21366 cDNA, wherein SEQ ID NO:361 is a clone designatedherein as “DNA143276-2975”.

FIG. 362 shows the amino acid sequence (SEQ ID NO:362) derived from thecoding sequence of SEQ ID NO:361 shown in FIG. 361.

FIG. 363 shows a nucleotide sequence (SEQ ID NO:363) of a nativesequence PRO20040 cDNA, wherein SEQ ID NO:363 is a clone designatedherein as “DNA164625-2890”.

FIG. 364 shows the amino acid sequence (SEQ ID NO:364) derived from thecoding sequence of SEQ ID NO:363 shown in FIG. 363.

FIG. 365 shows a nucleotide sequence (SEQ ID NO:365) of a nativesequence PRO21184 cDNA, wherein SEQ ID NO:365 is a clone designatedherein as “DNA167678-2963”.

FIG. 366 shows the amino acid sequence (SEQ ID NO:366) derived from thecoding sequence of SEQ ID NO:365 shown in FIG. 365.

FIG. 367 shows a nucleotide sequence (SEQ ID NO:367) of a nativesequence PRO21055 cDNA, wherein SEQ ID NO:367 is a clone designatedherein as “DNA170021-2923”.

FIG. 368 shows the amino acid sequence (SEQ ID NO:368) derived from thecoding sequence of SEQ ID NO:367 shown in FIG. 367.

FIG. 369 shows a nucleotide sequence (SEQ ID NO:369) of a nativesequence PRO28631 cDNA, wherein SEQ ID NO:369 is a clone designatedherein as “DNA170212-3000”.

FIG. 370 shows the amino acid sequence (SEQ ID NO:370) derived from thecoding sequence of SEQ ID NO:369 shown in FIG. 369.

FIG. 371 shows a nucleotide sequence (SEQ ID NO:371) of a nativesequence PRO21384 cDNA, wherein SEQ ID NO:371 is a clone designatedherein as “DNA177313-2982”.

FIG. 372 shows the amino acid sequence (SEQ ID NO:372) derived from thecoding sequence of SEQ ID NO:371 shown in FIG. 371.

FIG. 373 shows a nucleotide sequence (SEQ ID NO:373) of a nativesequence PRO1449 cDNA, wherein SEQ ID NO:373 is a clone designatedherein as “DNA64908-1163-1”.

FIG. 374 shows the amino acid sequence (SEQ ID NO:374) derived from thecoding sequence of SEQ ID NO:373 shown in FIG. 373.

FIG. 375 shows wholemount in situ hybridization results on mouse embryosusing a mouse orthologue of PRO1449 which has about 78% amino acididentity with PRO1449. The results show that PRO1449 orthologue isexpressed in the developing vasculature. The cross-section further showsexpression in endothelial cells and progenitors of endothelial cells.

FIG. 376 shows that a PRO1449 orthologue having about 78% amino acididentity with PRO1449 is expressed in vasculature of many inflamed anddiseased tissues, but is very low, or lacking, in normal adult vessels.

FIG. 377 shows that a PRO1449 orthologue having about 78% amino acididentity with PRO1449 induces ectopic vessels in the eyes of chickenembryos.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. Definitions

The phrases “cardiovascular, endothelial and angiogenic disorder”,“cardiovascular, endothelial and angiogenic dysfunction”,“cardiovascular, endothelial or angiogenic disorder” and“cardiovascular, endothelial or angiogenic dysfunction” are usedinterchangeably and refer in part to systemic disorders that affectvessels, such as diabetes mellitus, as well as diseases of the vesselsthemselves, such as of the arteries, capillaries, veins, and/orlymphatics. This would include indications that stimulate angiogenesisand/or cardiovascularization, and those that inhibit angiogenesis and/orcardiovascularization. Such disorders include, for example, arterialdisease, such as atherosclerosis, hypertension, inflammatoryvasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; and other vasculardisorders such as peripheral vascular disease, cancer such as vasculartumors, e.g., hemangioma (capillary and cavernous), glomus tumors,telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma, tumor angiogenesis, trauma such as wounds, burns, andother injured tissue, implant fixation, scarring, ischemia reperfusioninjury, rheumatoid arthritis, cerebrovascular disease, renal diseasessuch as acute renal failure, and osteoporosis. This would also includeangina, myocardial infarctions such as acute myocardial infarctions,cardiac hypertrophy, and heart failure such as CHF.

“Hypertrophy”, as used herein, is defined as an increase in mass of anorgan or structure independent of natural growth that does not involvetumor formation. Hypertrophy of an organ or tissue is due either to anincrease in the mass of the individual cells (true hypertrophy), or toan increase in the number of cells making up the tissue (hyperplasia),or both. Certain organs, such as the heart, lose the ability to divideshortly after birth. Accordingly, “cardiac hypertrophy” is defined as anincrease in mass of the heart, which, in adults, is characterized by anincrease in myocyte cell size and contractile protein content withoutconcomitant cell division. The character of the stress responsible forinciting the hypertrophy, (e.g., increased preload, increased afterload,loss of myocytes, as in myocardial infarction, or primary depression ofcontractility), appears to play a critical role in determining thenature of the response. The early stage of cardiac hypertrophy isusually characterized morphologically by increases in the size ofmyofibrils and mitochondria, as well as by enlargement of mitochondriaand nuclei. At this stage, while muscle cells are larger than normal,cellular organization is largely preserved. At a more advanced stage ofcardiac hypertrophy, there are preferential increases in the size ornumber of specific organelles, such as mitochondria, and new contractileelements are added in localized areas of the cells, in an irregularmanner. Cells subjected to long-standing hypertrophy show more obviousdisruptions in cellular organization, including markedly enlarged nucleiwith highly lobulated membranes, which displace adjacent myofibrils andcause breakdown of normal Z-band registration. The phrase “cardiachypertrophy” is used to include all stages of the progression of thiscondition, characterized by various degrees of structural damage of theheart muscle, regardless of the underlying cardiac disorder. Hence, theterm also includes physiological conditions instrumental in thedevelopment of cardiac hypertrophy, such as elevated blood pressure,aortic stenosis, or myocardial infarction.

“Heart failure” refers to an abnormality of cardiac function where theheart does not pump blood at the rate needed for the requirements ofmetabolizing tissues. The heart failure can be caused by a number offactors, including ischemic, congenital, rheumatic, or idiopathic forms.

“Congestive heart failure” (CHF) is a progressive pathologic state wherethe heart is increasingly unable to supply adequate cardiac output (thevolume of blood pumped by the heart over time) to deliver the oxygenatedblood to peripheral tissues. As CHF progresses, structural andhemodynamic damages occur. While these damages have a variety ofmanifestations, one characteristic symptom is ventricular hypertrophy.CHF is a common end result of a number of various cardiac disorders.

“Myocardial infarction” generally results from atherosclerosis of thecoronary arteries, often with superimposed coronary thrombosis. It maybe divided into two major types: transmural infarcts, in whichmyocardial necrosis involves the full thickness of the ventricular wall,and subendocardial (nontransmural) infarcts, in which the necrosisinvolves the subendocardium, the intramural myocardium, or both, withoutextending all the way through the ventricular wall to the epicardium.Myocardial infarction is known to cause both a change in hemodynamiceffects and an alteration in structure in the damaged and healthy zonesof the heart. Thus, for example, myocardial infarction reduces themaximum cardiac output and the stroke volume of the heart. Alsoassociated with myocardial infarction is a stimulation of the DNAsynthesis occurring in the interstice as well as an increase in theformation of collagen in the areas of the heart not affected.

As a result of the increased stress or strain placed on the heart inprolonged hypertension due, for example, to the increased totalperipheral resistance, cardiac hypertrophy has long been associated with“hypertension”. A characteristic of the ventricle that becomeshypertrophic as a result of chronic pressure overload is an impaireddiastolic performance. Fouad et al., J. Am. Coll. Cardiol., 4: 1500–1506(1984); Smith et al., J. Am. Coll. Cardiol., 5: 869–874 (1985). Aprolonged left ventricular relaxation has been detected in earlyessential hypertension, in spite of normal or supranormal systolicfunction. Hartford et al., Hypertension, 6: 329–338 (1984). However,there is no close parallelism between blood pressure levels and cardiachypertrophy. Although improvement in left ventricular function inresponse to antihypertensive therapy has been reported in humans,patients variously treated with a diuretic (hydrochlorothiazide), aβ-blocker (propranolol), or a calcium channel blocker (diltiazem), haveshown reversal of left ventricular hypertrophy, without improvement indiastolic function. Inouye et al, Am. J. Cardiol., 53: 1583–7 (1984).

Another complex cardiac disease associated with cardiac hypertrophy is“hypertrophic cardiomyopathy”. This condition is characterized by agreat diversity of morphologic, functional, and clinical features (Maronet al., N. Engl. J. Med., 316: 780–789 (1987); Spirito et al., N. Engl.J. Med., 320: 749–755 (1989); Louie and Edwards, Prog. Cardiovasc. Dis.,36: 275–308 (1994); Wigle et al., Circulation, 92: 1680–1692 (1995)),the heterogeneity of which is accentuated by the fact that it afflictspatients of all ages. Spirito et al., N. Engl. J. Med., 336: 775–785(1997). The causative factors of hypertrophic cardiomyopathy are alsodiverse and little understood. In general, mutations in genes encodingsarcomeric proteins are associated with hypertrophic cardiomyopathy.Recent data suggest that β-myosin heavy chain mutations may account forapproximately 30 to 40 percent of cases of familial hypertrophiccardiomyopathy. Watkins et al., N. Engl. J. Med., 326: 1108–1114 (1992);Schwartz et al, Circulation, 91: 532–540 (1995); Marian and Roberts,Circulation, 92: 1336–1347 (1995); Thierfelder et al., Cell, 77: 701–712(1994); Watkins et al., Nat. Gen., 11: 434–437 (1995). Besides β-myosinheavy chain, other locations of genetic mutations include cardiactroponin T, alpha topomyosin, cardiac myosin binding protein C,essential myosin light chain, and regulatory myosin light chain. See,Malik and Watkins, Curr. Opin. Cardiol., 12: 295–302 (1997).

Supravalvular “aortic stenosis” is an inherited vascular disordercharacterized by narrowing of the ascending aorta, but other arteries,including the pulmonary arteries, may also be affected. Untreated aorticstenosis may lead to increased intracardiac pressure resulting inmyocardial hypertrophy and eventually heart failure and death. Thepathogenesis of this disorder is not fully understood, but hypertrophyand possibly hyperplasia of medial smooth muscle are prominent featuresof this disorder. It has been reported that molecular variants of theelastin gene are involved in the development and pathogenesis of aorticstenosis. U.S. Pat. No. 5,650,282 issued Jul. 22, 1997.

“Valvular regurgitation” occurs as a result of heart diseases resultingin disorders of the cardiac valves. Various diseases, like rheumaticfever, can cause the shrinking or pulling apart of the valve orifice,while other diseases may result in endocarditis, an inflammation of theendocardium or lining membrane of the atrioventricular orifices andoperation of the heart. Defects such as the narrowing of the valvestenosis or the defective closing of the valve result in an accumulationof blood in the heart cavity or regurgitation of blood past the valve.If uncorrected, prolonged valvular stenosis or insufficiency may resultin cardiac hypertrophy and associated damage to the heart muscle, whichmay eventually necessitate valve replacement.

The treatment of all these, and other cardiovascular, endothelial andangiogenic disorders, which may or may not be accompanied by cardiachypertrophy, is encompassed by the present invention.

The terms “cancer”, “cancerous”, and “malignant” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include but are notlimited to, carcinoma including adenocarcinoma, lymphoma, blastoma,melanoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin'slymphoma, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer such as hepatic carcinoma and hepatoma, bladdercancer, breast cancer, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer such as renal cellcarcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, and various types of head and neck cancer. The preferred cancersfor treatment herein are breast, colon, lung, melanoma, ovarian, andothers involving vascular tumors as noted above.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., ¹³¹I,¹²⁵I, ⁹⁰Y, and ¹⁸⁶Re), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant, or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents, folic acid antagonists, anti-metabolites of nucleicacid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil,cisplatin, purine nucleosides, amines, amino acids, triazol nucleosides,or corticosteroids. Specific examples include Adriamycin, Doxorubicin,5-Fluorouracil, Cytosine arabinoside (“Ara-C”), Cyclophosphamide,Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin,Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan, and other relatednitrogen mustards. Also included in this definition are hormonal agentsthat act to regulate or inhibit hormone action on tumors, such astamoxifen and onapristone.

A “growth-inhibitory agent” when used herein refers to a compound orcomposition that inhibits growth of a cell, such as anWnt-overexpressing cancer cell, either in vitro or in vivo. Thus, thegrowth-inhibitory agent is one which significantly reduces thepercentage of malignant cells in S phase. Examples of growth-inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxol, and topo II inhibitors such as doxorubicin,daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 alsospill over into S-phase arrest, for example, DNA alkylating agents suchas tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in The Molecular Basis of Cancer, Mendelsohn and Israel, eds.,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (WB Saunders: Philadelphia,1995), especially p. 13. Additional examples include tumor necrosisfactor (TNF), an antibody capable of inhibiting or neutralizing theangiogenic activity of acidic or basic FGF or hepatocyte growth factor(HGF), an antibody capable of inhibiting or neutralizing the coagulantactivities of tissue factor, protein C, or protein S (see, WO 91/01753,published Feb. 21, 1991), or an antibody capable of binding to HER2receptor (WO 89/06692), such as the 4D5 antibody (and functionalequivalents thereof) (e.g., WO 92/22653).

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of acardiovascular, endothelial, and angiogenic disorder. The concept oftreatment is used in the broadest sense, and specifically includes theprevention (prophylaxis), moderation, reduction, and curing ofcardiovascular, endothelial, and angiogenic disorders of any stage.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) or ameliorate a cardiovascular, endothelial, andangiogenic disorder such as hypertrophy. Those in need of treatmentinclude those already with the disorder as well as those prone to havethe disorder or those in whom the disorder is to be prevented. Thedisorder may result from any cause, including idiopathic, cardiotrophic,or myotrophic causes, or ischemia or ischemic insults, such asmyocardial infarction.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial effect, such as an anti-hypertrophic effect, for an extendedperiod of time.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, pigs, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The phrase “cardiovascular, endothelial or angiogenic agents” refersgenerically to any drug that acts in treating cardiovascular,endothelial, and angiogenic disorders. Examples of cardiovascular agentsare those that promote vascular homeostasis by modulating bloodpressure, heart rate, heart contractility, and endothelial and smoothmuscle biology, all of which factors have a role in cardiovasculardisease. Specific examples of these include angiotensin-II receptorantagonists; endothelin receptor antagonists such as, for example,BOSENTAN™ and MOXONODIN™; interferon-gamma (IFN-γ);des-aspartate-angiotensin I; thrombolytic agents, e.g., streptokinase,urokinase, t-PA, and a t-PA variant specifically designed to have longerhalf-life and very high fibrin specificity, TNK t-PA (a T103N, N117Q,KHRR(296–299)AAAA t-PA variant, Keyt et al., Proc. Natl. Acad. Sci. USA,91: 3670–3674 (1994)); inotropic or hypertensive agents such asdigoxigenin and β-adrenergic receptor blocking agents, e.g.,propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol,penbutolol, acetobutolol, atenolol, metoprolol, and carvedilol;angiotensin converting enzyme (ACE) inhibitors, e.g., quinapril,captopril, enalapril, ramipril, benazepril, fosinopril, and lisinopril;diuretics, e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, andindapamide; and calcium channel blockers, e.g., diltiazem, nifedipine,verapamil, nicardipine. One preferred category of this type is atherapeutic agent used for the treatment of cardiac hypertrophy or of aphysiological condition instrumental in the development of cardiachypertrophy, such as elevated blood pressure, aortic stenosis, ormyocardial infarction.

“Angiogenic agents” and “endothelial agents” are active agents thatpromote angiogenesis and/or endothelial cell growth, or, if applicable,vasculogenesis. This would include factors that accelerate woundhealing, such as growth hormone, insulin-like growth factor-I (IGF-I),VEGF, VIGF, PDGF, epidermal growth factor (EGF), CTGF and members of itsfamily, FGF, and TGF-α and TGF-β.

“Angiostatic agents” are active agents that inhibit angiogenesis orvasculogenesis or otherwise inhibit or prevent growth of cancer cells.Examples include antibodies or other antagonists to angiogenic agents asdefined above, such as antibodies to VEGF. They additionally includecytotherapeutic agents such as cytotoxic agents, chemotherapeuticagents, growth-inhibitory agents, apoptotic agents, and other agents totreat cancer, such as anti-HER-2, anti-CD20, and other bioactive andorganic chemical agents.

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” of an active agent such as a PROpolypeptide or agonist or antagonist thereto or an anti-PRO antibody,refers to an amount effective in the treatment of a cardiovascular,endothelial or angiogenic disorder in a mammal and can be determinedempirically.

As used herein, an “effective amount” of an active agent such as a PROpolypeptide or agonist or antagonist thereto or an anti-PRO antibody,refers to an amount effective for carrying out a stated purpose, whereinsuch amounts may be determined empirically for the desired effect.

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

A “native sequence PRO polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding PRO polypeptide derivedfrom nature. Such native sequence PRO polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence PRO polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific PROpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

The PRO polypeptide “extracellular domain” or “ECD” refers to a form ofthe PRO polypeptide which is essentially free of the transmembrane andcytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have lessthan 1% of such transmembrane and/or cytoplasmic domains and preferably,will have less than 0.5% of such domains. It will be understood that anytransmembrane domains identified for the PRO polypeptides of the presentinvention are identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified herein. Optionally, therefore, an extracellular domain of aPRO polypeptide may contain from about 5 or fewer amino acids on eitherside of the transmembrane domain/extracellular domain boundary asidentified in the Examples or specification and such polypeptides, withor without the associated signal peptide, and nucleic acid encodingthem, are comtemplated by the present invention.

The approximate location of the “signal peptides” of the various PROpolypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng., 10:1–6(1997) and von Heinje et al., Nucl. Acids Res., 14:4683–4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“PRO polypeptide variant” means an active PRO polypeptide as definedabove or below having at least about 80% amino acid sequence identitywith a full-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or98% amino acid sequence identity and alternatively at least about 99%amino acid sequence identity to a full-length native sequence PROpolypeptide sequence as disclosed herein, a PRO polypeptide sequencelacking the signal peptide as disclosed herein, an extracellular domainof a PRO polypeptide, with or without the signal peptide, as disclosedherein or any other specifically defined fragment of a full-length PROpolypeptide sequence as disclosed herein. Ordinarily, PRO variantpolypeptides are at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150 or 200 amino acids in length and alternatively at least about 300amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the PROpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in a PRO sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full-length of the sequences beingcompared. For purposes herein, however, % amino acid sequence identityvalues are obtained as described below by using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is provided in Table 1. The ALIGN-2 sequence comparisoncomputer program was authored by Genentech, Inc., and the source codeshown in Table 1 has been filed with user documentation in the U.S.Copyright Office, Washington D.C., 20559, where it is registered underU.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available through Genentech, Inc., South San Francisco, Calif.or may be compiled from the source code provided in Table 1. The ALIGN-2program should be compiled for use on a UNIX operating system,preferably digital UNIX V4.0D. All sequence comparison parameters areset by the ALIGN-2 program and do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program'alignment of Aand B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations, Tables 2–3 demonstrate how to calculate the % amino acidsequence identity of the amino acid sequence designated “ComparisonProtein” to the amino acid sequence designated “PRO”.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described above using the ALIGN-2sequence comparison computer program. However, % amino acid sequenceidentity may also be determined using the sequence comparison programNCBI-BLAST2 (Altschul et al., Nucleic Acids Res., 25:3389–3402 (1997)).The NCBI-BLAST2 sequence comparison program may be obtained from theNational Institute of Health, Bethesda, Md. NCBI-BLAST2 uses severalsearch parameters, wherein all of those search parameters are set todefault values including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

In addition, % amino acid sequence identity may also be determined usingthe WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology,266:460–480 (1996)). Most of the WU-BLAST-2 search parameters are set tothe default values. Those not set to default values, i.e., theadjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. For purposes herein, a % amino acid sequence identityvalue is determined by dividing (a) the number of matching identicalamino acids residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

“PRO variant polynucleotide” or “PRO variant nucleic acid sequence”means a nucleic acid molecule which encodes an active PRO polypeptide asdefined below and which has at least about 80% nucleic acid sequenceidentity with a nucleotide acid sequence encoding a full-length nativesequence PRO polypeptide sequence as disclosed herein, a full-lengthnative sequence PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Ordinarily,a PRO variant polynucleotide will have at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97% or 98% nucleic acid sequence identity and alternatively at leastabout 99% nucleic acid sequence identity with a nucleic acid sequenceencoding a full-length native sequence PRO polypeptide sequence asdisclosed herein, a full-length native sequence PRO polypeptide sequencelacking the signal peptide as disclosed herein, an extracellular domainof a PRO polypeptide, with or without the signal sequence, as disclosedherein or any other fragment of a full-length PRO polypeptide sequenceas disclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, PRO variant polynucleotides are at least about 30, 60, 90,120, 150, 180, 210, 240, 270, 300, 450, or 600 nucleotides in length andalternatively at least about 900 nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect to the PROpolypeptide-encoding nucleic acid sequences identified herein is definedas the percentage of nucleotides in a candidate sequence that areidentical with the nucleotides in a PRO polypeptide-encoding nucleicacid sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent nucleic acid sequence identity canbe achieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % nucleic acid sequence identity values are obtained asdescribed below by using the sequence comparison computer programALIGN-2, wherein the complete source code for the ALIGN-2 program isprovided in Table 1. The ALIGN-2 sequence comparison computer programwas authored by Genentech, Inc., and the source code shown in Table 1has been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in Table 1. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % nucleic acid sequence identity of a givennucleic acid sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given nucleic acidsequence C that has or comprises a certain % nucleic acid sequenceidentity to, with, or against a given nucleic acid sequence D) iscalculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4–5 demonstrate how to calculate the % nucleic acidsequence identity of the nucleic acid sequence designated “ComparisonDNA” to the nucleic acid sequence designated “PRO-DNA”.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described above using theALIGN-2 sequence comparison computer program. However, % nucleic acidsequence identity may also be determined using the sequence comparisonprogram NCBI-BLAST2 (Altschul et al., Nucleic Acids Res., 25:3389–3402(1997)). The NCBI-BLAST2 sequence comparison program may be obtainedfrom the National Institute of Health, Bethesda, Md. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In addition, % nucleic acid sequence identity values may also begenerated using the WU-BLAST-2 computer program (Altschul et al.,Methods in Enzymology, 266:460–480 (1996)). Most of the WU-BLAST-2search parameters are set to the default values. Those not set todefault values, i.e., the adjustable parameters, are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11, and scoring matrix=BLOSUM62. For purposes herein, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

In other embodiments, PRO variant polynucleotides are nucleic acidmolecules that encode an active PRO polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding the full-length PROpolypeptide as shown in the specification herein and accompanyingfigures. PRO variant polypeptides may be those that are encoded by a PROvariant polynucleotide.

“Isolated”, when used to describe the various polypeptides disclosedherein, means a polypeptide that has been identified and separatedand/or recovered from a component of its natural environment.Preferably, the isolated polypeptide is free of association with allcomponents with which it is naturally associated. Contaminant componentsof its natural environment are materials that would typically interferewith diagnostic or therapeutic uses for the polypeptide, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the polypeptide will be purified (1) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (2)to homogeneity by SDS-PAGE under non-reducing or reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated polypeptideincludes polypeptide in situ within recombinant cells, since at leastone component of the PRO natural environment will not be present.Ordinarily, however, isolated polypeptide will be prepared by at leastone purification step.

An “isolated” nucleic acid molecule encoding a PRO polypeptide or an“isolated” nucleic acid molecule encoding an anti-PRO antibody is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the PRO-encoding nucleic acid or the naturalsource of the anti-PRO-encoding nucleic acid. Preferably, the isolatednucleic acid is free of association with all components with which it isnaturally associated. An isolated PRO-encoding nucleic acid molecule oran isolated anti-PRO-encoding nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the PRO-encoding nucleic acidmolecule or from the anti-PRO-encoding nucleic acid molecule as itexists in natural cells. However, an isolated nucleic acid moleculeencoding a PRO polypeptide or an isolated nucleic acid molecule encodingan anti-PRO antibody includes PRO-nucleic acid molecules oranti-PRO-nucleic acid molecules contained in cells that ordinarilyexpress PRO polypeptides or anti-PRO antibodies where, for example, thenucleic acid molecule is in a chromosomal location different from thatof natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize, forexample, promoters, polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for a PROpolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in the same reading frame.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see, Ausubel etal., Current Protocols in Molecular Biology (Wiley IntersciencePublishers, 1995).

“Stringent conditions” or “high-stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example, 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately-stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Press, 1989), and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength, and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37–50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The modifier “epitope-tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

“Active” or “activity” in the context of PRO variants refers to form(s)of PRO proteins that retain the biologic and/or immunologic activitiesof a native or naturally-occurring PRO polypeptide.

“Biological activity” in the context of a molecule that antagonizes aPRO polypeptide that can be identified by the screening assays disclosedherein (e.g., an organic or inorganic small molecule, peptide, etc.) isused to refer to the ability of such molecules to bind or complex withthe PRO polypeptide identified herein, or otherwise interfere with theinteraction of the PRO polypeptide with other cellular proteins orotherwise inhibits the transcription or translation of the PROpolypeptide. Particularly preferred biological activity includes cardiachypertrophy, activity that acts on systemic disorders that affectvessels, such as diabetes mellitus, as well as diseases of the arteries,capillaries, veins, and/or lymphatics, and cancer.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes one ormore of the biological activities of a native PRO polypeptide disclosedherein, for example, if applicable, its mitogenic or angiogenicactivity. Antagonists of a PRO polypeptide may act by interfering withthe binding of a PRO polypeptide to a cellular receptor, byincapacitating or killing cells that have been activated by a PROpolypeptide, or by interfering with vascular endothelial cell activationafter binding of a PRO polypeptide to a cellular receptor. All suchpoints of intervention by a PRO polypeptide antagonist shall beconsidered equivalent for purposes of this invention. The antagonistsinhibit the mitogenic, angiogenic, or other biological activity of PROpolypeptides, and thus are useful for the treatment of diseases ordisorders characterized by undesirable excessive neovascularization,including by way of example tumors, and especially solid malignanttumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic andother retinopathies, retrolental fibroplasia, age-related maculardegeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias(including Grave's disease), corneal and other tissue transplantation,and chronic inflammation. The antagonists also are useful for thetreatment of diseases or disorders characterized by undesirableexcessive vascular permeability, such as edema associated with braintumors, ascites associated with malignancies, Meigs' syndrome, lunginflammation, nephrotic syndrome, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion. In a similarmanner, the term “agonist” is used in the broadest sense and includesany molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments, or amino acid sequence variants of native PROpolypeptides, peptides, small organic molecules, etc.

A “small molecule” is defined herein to have a molecular weight belowabout 500 daltons.

The term “PRO polypeptide receptor” as used herein refers to a cellularreceptor for a PRO polypeptide, ordinarily a cell-surface receptor foundon vascular endothelial cells, as well as variants thereof that retainthe ability to bind a PRO polypeptide.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.The term “antibody” is used in the broadest sense and specificallycovers, without limitation, intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies)formed from at least two intact antibodies, and antibody fragments, solong as they exhibit the desired biological activity.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody to andfor its particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three segments called complementarity-determiningregions (CDRs) or hypervariable regions both in the light-chain and theheavy-chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FR). The variabledomains of native heavy and light chains each comprise four FR regions,largely adopting a β-sheet configuration, connected by three CDRs, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen-binding site of antibodies. See, Kabat etal., NIH Publ. No. 91-3242, Vol. I, pages 647–669 (1991). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.,8(10): 1057–1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments that have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM; and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352: 624–628 (1991) and Marks et al., J. Mol. Biol., 222: 581–597(1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity. U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851–6855 (1984).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)₂, or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv FR residuesof the human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues thatare found neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andmaximize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody preferably also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature, 321:522–525 (1986); Reichmann et al., Nature, 332: 323–329 (1988); andPresta, Curr. Op. Struct. Biol., 2: 593–596 (1992). The humanizedantibody includes a PRIMATIZED™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv see, Pluckthun in The Pharmacology of MonoclonalAntibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag: NewYork, 1994), pp. 269–315.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90: 6444–6448 (1993).

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells, since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

The word “label” when used herein refers to a detectable compound orother composition that is conjugated directly or indirectly to theantibody so as to generate a “labeled” antibody. The label may bedetectable by itself (e.g., radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition that is detectable. Radionuclidesthat can serve as detectable labels include, for example, I-131, I-123,I-125, Y-90, Re-188, At-211, Cu-67, Bi-212, and Pd-109. The label mayalso be a non-detectable entity such as a toxin.

By “solid phase” is meant a non-aqueous matrix to which an antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant that is useful for delivery of a drug(such as the PRO polypeptide or antibodies thereto disclosed herein) toa mammal. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

As used herein, the term “immunoadhesin” designates antibody-likemolecules that combine the binding specificity of a heterologous protein(an “adhesin”) with the effector functions of immunoglobulin constantdomains. Structurally, the immunoadhesins comprise a fusion of an aminoacid sequence with the desired binding specificity that is other thanthe antigen recognition and binding site of an antibody (i.e., is“heterologous”), and an immunoglobulin constant domain sequence. Theadhesin part of an immunoadhesin molecule typically is a contiguousamino acid sequence comprising at least the binding site of a receptoror a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD, or IgM.

As shown below, Table 1 provides the complete source code for theALIGN-2 sequence comparison computer program. This source code may beroutinely compiled for use on a UNIX operating system to provide theALIGN-2 sequence comparison computer program.

In addition, Tables 2–5 show hypothetical exemplifications for using thebelow described method to determine % amino acid sequence identity(Tables 2–3) and % nucleic acid sequence identity (Tables 4–5) using theALIGN-2 sequence comparison computer program, wherein “PRO” representsthe amino acid sequence of a hypothetical PRO polypeptide of interest,“Comparison Protein” represents the amino acid sequence of a polypeptideagainst which the “PRO” polypeptide of interest is being compared,“PRO-DNA” represents a hypothetical PRO-encoding nucleic acid sequenceof interest, “Comparison DNA” represents the nucleotide sequence of anucleic acid molecule against which the “PRO-DNA” nucleic acid moleculeof interest is being compared, “X”, “Y”, and “Z” each representdifferent hypothetical amino acid residues and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity =(the number of identically matching nucleotides between the two nucleicacid sequences as determined by ALIGN-2) divided by (the total number ofnucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 =33.3%

5.2. Compositions and Methods of the Invention

5.2.1. PRO Variants

In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO polypeptidesuch as changing the number or position of glycosylation sites oraltering the membrane anchoring characteristics.

Variations in the native full-length sequence PRO polypeptide or invarious domains of the PRO polypeptide described herein, can be made,for example, using any of the techniques and guidelines for conservativeand non-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the PRO polypeptide that results in a changein the amino acid sequence of the PRO polypeptide as compared with thenative sequence PRO polypetide. Optionally the variation is bysubstitution of at least one amino acid with any other amino acid in oneor more of the domains of the PRO polypeptide. Guidance in determiningwhich amino acid residue may be inserted, substituted or deleted withoutadversely affecting the desired activity may be found by comparing thesequence of the PRO polypeptide with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thePRO polypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, glu;-   (4) basic: asn, gln, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081–1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

5.2.2. Modifications of PRO Polypeptides

Covalent modifications of PRO polypeptides are included within the scopeof this invention. One type of covalent modification includes reactingtargeted amino acid residues of a PRO polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C- terminal residues of the PRO polypeptide. Derivatizationwith bifunctional agents is useful, for instance, for crosslinking thePRO polypeptide to a water-insoluble support matrix or surface for usein the method for purifying anti-PRO antibodies, and vice-versa.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79–86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in the native sequence PROpolypeptide (either by removing the underlying glycosylation site or bydeleting the glycosylation by chemical and/or enzymatic means), and/oradding one or more glycosylation sites that are not present in thenative sequence PRO polypeptide. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO polypeptide (forO-linked glycosylation sites). The PRO amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the PRO polypeptide at preselected bases suchthat codons are generated that will translate into the desired aminoacids.

Another means of increasing the number of carbohydrate moieties on thePRO polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259–306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of the PRO polypeptide compriseslinking the PRO polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PRO polypeptide of the present invention may also be modified in away to form a chimeric molecule comprising the PRO polypeptide fused toanother, heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO polypeptide with a protein transduction domain which targets the PROpolypeptide for delivery to various tissues and more particularly acrossthe brain blood barrier, using, for example, the protein transductiondomain of human immunodeficiency virus TAT protein (Schwarze et al.,1999, Science 285: 1569–72).

In another embodiment, such a chimeric molecule comprises a fusion ofthe PRO polypeptide with a tag polypeptide which provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino- or carboxyl-terminus of the PROpolypeptide. The presence of such epitope-tagged forms of the PROpolypeptide can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables the PROpolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-His) orpoly-histidine-glycine (poly-His-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159–2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610–3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547–553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204–1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192–194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163–15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393–6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO polypeptide with an immunoglobulin or a particularregion of an immunoglobulin. For a bivalent form of the chimericmolecule (also referred to as an “immunoadhesin”), such a fusion couldbe to the Fc region of an IgG molecule. The Ig fusions preferablyinclude the substitution of a soluble (transmembrane domain deleted orinactivated) form of a PRO polypeptide in place of at least one variableregion within an Ig molecule. In a particularly preferred embodiment,the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also, U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

5.2.3. Preparation of the PRO Polypeptide

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO polypeptides. In particular, cDNAs encoding PRO polypeptides havebeen identified and isolated, as disclosed in further detail in theExamples below. It is noted that proteins produced in separateexpression rounds may be given different PRO numbers but the UNQ numberis unique for any given DNA and the encoded protein, and will not bechanged. However, for sake of simplicity, in the present specificationthe protein encoded by the PRO DNA as well as all further nativehomologues and variants included in the foregoing definition of PROpolypeptides, will be referred to as “PRO” regardless of their origin ormode of preparation.

The description below relates primarily to production of PROpolypeptides by culturing cells transformed or transfected with a vectorcontaining nucleic acid encoding PRO polypeptides. It is, of course,contemplated that alternative methods that are well known in the art maybe employed to prepare the PRO polypeptide. For instance, the PROpolypeptide sequence, or portions thereof, may be produced by directpeptide synthesis using solid-phase techniques. See, e.g., Stewart etal., Solid-Phase Peptide Synthesis (W.H. Freeman Co.: San Francisco,Calif., 1969); Merrifield, J. Am. Chem. Soc., 85: 2149–2154 (1963). Invitro protein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be accomplished, for instance, withan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of the PRO polypeptide maybe chemically synthesized separately and combined using chemical orenzymatic methods to produce the full-length PRO polypeptide.

5.2.3.1. Isolation of DNA Encoding PRO Polypeptides

DNA encoding the PRO polypeptide may be obtained from a cDNA libraryprepared from tissue believed to possess the mRNA encoding the PROpolypeptide and to express it at a detectable level. Accordingly, DNAsencoding the human PRO polypeptide can be conveniently obtained fromcDNA libraries prepared from human tissues, such as described in theExamples. The gene encoding the PRO polypeptide may also be obtainedfrom a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the PROpolypeptide or oligonucleotides of at least about 20–80 bases) designedto identify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al., supra.An alternative means to isolate the gene encoding the PRO polypeptide isto use PCR methodology. Sambrook et al., supra; Dieffenbach et al., PCRPrimer: A Laboratory Manual (New York: Cold Spring Harbor LaboratoryPress, 1995).

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation, or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as ALIGN, DNAstar, and INHERIT, which employ various algorithms tomeasure homology.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

5.2.3.2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO polypeptide production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH, and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ treatment and electroporation. Depending on the host cellused, transformation is performed using standard techniques appropriateto such cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23: 315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456–457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130: 946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene or polyornithine, may also beused. For various techniques for transforming mammalian cells, see,Keown et al., Methods in Enzymology, 185: 527–537 (1990) and Mansour etal., Nature, 336: 348–352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include, but are not limited to, eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325); and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for vectorsencoding the PRO polypeptide. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published May 2, 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9: 968–975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8: 135(1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,28: 265–278 [1988]); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259–5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published Oct. 31, 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published Jan.10, 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112: 284–289 [1983]; Tilburn et al.,Gene, 26: 205–221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470–1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4: 475–479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of nucleic acid encodingglycosylated PRO polypeptides are derived from multicellular organisms.Examples of invertebrate cells include insect cells such as DrosophilaS2 and Spodoptera Sf9, as well as plant cells. Examples of usefulmammalian host cell lines include Chinese hamster ovary (CHO) and COScells. More specific examples include monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. Gen.Virol., 36: 59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlauband Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertolicells (TM4, Mather, Biol. Reprod., 23:243–251 (1980)); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mousemammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriatehost cell is deemed to be within the skill in the art.

5.2.3.3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the PROpolypeptide may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence if thesequence is to be secreted, an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques that are known tothe skilled artisan.

The PRO polypeptide may be produced recombinantly not only directly, butalso as a fusion polypeptide with a heterologous polypeptide, which maybe a signal sequence or other polypeptide having a specific cleavagesite at the N-terminus of the mature protein or polypeptide. In general,the signal sequence may be a component of the vector, or it may be apart of the DNA encoding the PRO polypeptide that is inserted into thevector. The signal sequence may be a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, 1pp, or heat-stable enterotoxin II leaders. For yeastsecretion the signal sequence may be, e.g., the yeast invertase leader,alpha factor leader (including Saccharomyces and Kluyveromyces α-factorleaders, the latter described in U.S. Pat. No. 5,010,182), or acidphosphatase leader, the C. albicans glucoamylase leader (EP 362,179published Apr. 4, 1990), or the signal described in WO 90/13646published Nov. 15, 1990. In mammalian cell expression, mammalian signalsequences may be used to direct secretion of the protein, such as signalsequences from secreted polypeptides of the same or related species, aswell as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV, or BPV) areuseful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the nucleicacid encoding the PRO polypeptide such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). A suitableselection gene for use in yeast is the trp1gene present in the yeastplasmid YRp7. Stinchcomb et al., Nature, 282: 39 (1979); Kingsman etal., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85: 12 (1977).

Expression and cloning vectors usually contain a promoter operablylinked to the nucleic acid sequence encoding the PRO polypeptide todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known. Promoters suitable for use with prokaryotichosts include the β-lactamase and lactose promoter systems (Chang etal., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)),alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel,Nucleic Acids Res., 8: 4057 (1980); EP 36,776), and hybrid promoterssuch as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21–25 (1983)). Promoters for use in bacterial systems also will containa Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding thePRO polypeptide.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17: 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters that are inducible promoters having the additionaladvantage of transcription controlled by growth conditions are thepromoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,metallothlionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in EP73,657.

PRO nucleic acid transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5,Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus,and Simian Virus 40 (SV40); by heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter; and by heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the PRO polypeptide by higher eukaryotesmay be increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, that act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100–270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thesequence coding for PRO polypeptides, but is preferably located at asite 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the PRO polypeptide.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of the PRO polypeptide in recombinant vertebrate cellculture are described in Gething et al., Nature, 293: 620–625 (1981);Mantei et al., Nature, 281: 40–46(1979); EP 117,060; and EP 117,058.

5.2.3.4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201–5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native-sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to DNAencoding the PRO polypeptide and encoding a specific antibody epitope.

5.2.3.5. Purification of PRO Polypeptides

Forms of PRO polypeptides may be recovered from culture medium or fromhost cell lysates. If membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g., TRITON-X™ 100) or byenzymatic cleavage. Cells employed in expression of nucleic acidencoding the PRO polypeptide can be disrupted by various physical orchemical means, such as freeze-thaw cycling, sonication, mechanicaldisruption, or cell-lysing agents. It may be desired to purify the PROpolypeptide from recombinant cell proteins or polypeptides. Thefollowing procedures are exemplary of suitable purification procedures:by fractionation on an ion-exchange column; ethanol precipitation;reverse phase HPLC; chromatography on silica or on a cation-exchangeresin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfateprecipitation; gel filtration using, for example, Sephadex G-75; proteinA Sepharose columns to remove contaminants such as IgG; and metalchelating columns to bind epitope-tagged forms of the PRO polypeptide.Various methods of protein purification may be employed and such methodsare known in the art and described, for example, in Deutscher, Methodsin Enzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice (Springer-Verlag: New York, 1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular PRO polypeptide produced.

5.2.4. Uses of PRO Polypeptides

5.2.4.1. Assays for Cardiovascular, Endothelial, and Angiogenic Activity

Various assays can be used to test the polypeptide herein forcardiovascular, endothelial, and angiogenic activity. Such assaysinclude those provided in the Examples below.

Assays for testing for endothelin antagonist activity, as disclosed inU.S. Pat. No. 5,773,414, include a rat heart ventricle binding assaywhere the polypeptide is tested for its ability to inhibit iodinizedendothelin-1 binding in a receptor assay, an endothelin receptor bindingassay testing for intact cell binding of radiolabeled endothelin-1 usingrabbit renal artery vascular smooth muscle cells, an inositol phosphateaccumulation assay where functional activity is determined in Rat-1cells by measuring intra-cellular levels of second messengers, anarachidonic acid release assay that measures the ability of addedcompounds to reduce endothelin-stimulated arachidonic acid release incultured vascular smooth muscles, in vitro (isolated vessel) studiesusing endothelium from male New Zealand rabbits, and in vivo studiesusing male Sprague-Dawley rats.

Assays for tissue generation activity include, without limitation, thosedescribed in WO 95/16035 (bone, cartilage, tendon); WO 95/05846 (nerve,neuronal), and WO 91/07491 (skin, endothelium).

Assays for wound-healing activity include, for example, those describedin Winter, Epidermal Wound Healing, Maibach, H I and Rovee, D T, eds.(Year Book Medical Publishers, Inc., Chicago), pp. 71–112, as modifiedby the article of Eaglstein and Mertz, J. Invest. Dermatol., 71: 382–384(1978).

An assay to screen for a test molecule relating to a PRO polypeptidethat binds an endothelin B₁ (ETB₁) receptor polypeptide and modulatessignal transduction activity involves providing a host cell transformedwith a DNA encoding endothelin B₁ receptor polypeptide, exposing thecells to the test candidate, and measuring endothelin B₁ receptor signaltransduction activity, as described, e.g., in U.S. Pat. No. 5,773,223.

There are several cardiac hypertrophy assays. In vitro assays includeinduction of spreading of adult rat cardiac myocytes. In this assay,ventricular myocytes are isolated from a single (male Sprague-Dawley)rat, essentially following a modification of the procedure described indetail by Piper et al., “Adult ventricular rat heart muscle cells” inCell Culture Techniques in Heart and Vessel Research, H. M. Piper, ed.(Berlin: Springer-Verlag, 1990), pp. 36–60. This procedure permits theisolation of adult ventricular myocytes and the long-term culture ofthese cells in the rod-shaped phenotype. Phenylephrine and ProstaglandinF_(2α) (PGF_(2α)) have been shown to induce a spreading response inthese adult cells. The inhibition of myocyte spreading induced byPGF_(2α) or PGF_(2α) analogs (e.g., fluprostenol) and phenylephrine byvarious potential inhibitors of cardiac hypertrophy is then tested.

One example of an in vivo assay is a test for inhibiting cardiachypertrophy induced by fluprostenol in vivo. This pharmacological modeltests the ability of the PRO polypeptide to inhibit cardiac hypertrophyinduced in rats (e.g., male Wistar or Sprague-Dawley) by subcutaneousinjection of fluprostenol (an agonist analog of PGF_(2α)). It is knownthat rats with pathologic cardiac hypertrophy induced by myocardialinfarction have chronically elevated levels of extractable PGF_(2α) intheir myocardium. Lai et al., Am. J. Physiol. (Heart Circ. Physiol.),271: H2197–H2208 (1996). Accordingly, factors that can inhibit theeffects of fluprostenol on myocardial growth in vivo are potentiallyuseful for treating cardiac hypertrophy. The effects of the PROpolypeptide on cardiac hypertrophy are determined by measuring theweight of heart, ventricles, and left ventricle (normalized by bodyweight) relative to fluprostenol-treated rats not receiving the PROpolypeptide.

Another example of an in vivo assay is the pressure-overload cardiachypertrophy assay. For in vivo testing it is common to inducepressure-overload cardiac hypertrophy by constriction of the abdominalaorta of test animals. In a typical protocol, rats (e.g., male Wistar orSprague-Dawley) are treated under anesthesia, and the abdominal aorta ofeach rat is narrowed down just below the diaphragm. Beznak M., Can. J.Biochem. Physiol., 33: 985–94 (1955). The aorta is exposed through asurgical incision, and a blunted needle is placed next to the vessel.The aorta is constricted with a ligature of silk thread around theneedle, which is immediately removed and which reduces the lumen of theaorta to the diameter of the needle. This approach is described, forexample, in Rossi et al., Am. Heart J., 124: 700–709 (1992) and O'Rourkeand Reibel, P.S.E.M.B., 200: 95–100 (1992).

In yet another in vivo assay, the effect on cardiac hypertrophyfollowing experimentally induced myocardial infarction (MI) is measured.Acute MI is induced in rats by left coronary artery ligation andconfirmed by electrocardiographic examination. A sham-operated group ofanimals is also prepared as control animals. Earlier data have shownthat cardiac hypertrophy is present in the group of animals with MI, asevidenced by an 18% increase in heart weight-to-body weight ratio. Laiet al., supra. Treatment of these animals with candidate blockers ofcardiac hypertrophy, e.g., the PRO polypeptide, provides valuableinformation about the therapeutic potential of the candidates tested.One further such assay test for induction of cardiac hypertrophy isdisclosed in U.S. Pat. No. 5,773,415, using Sprague-Dawley rats.

For cancer, a variety of well-known animal models can be used to furtherunderstand the role of the genes identified herein in the developmentand pathogenesis of tumors, and to test the efficacy of candidatetherapeutic agents, including antibodies and other antagonists of nativePRO polypeptides, such as small-molecule antagonists. The in vivo natureof such models makes them particularly predictive of responses in humanpatients. Animal models of tumors and cancers (e.g., breast cancer,colon cancer, prostate cancer, lung cancer, etc.) include bothnon-recombinant and recombinant (transgenic) animals. Non-recombinantanimal models include, for example, rodent, e.g., murine models. Suchmodels can be generated by introducing tumor cells into syngeneic miceusing standard techniques, e.g., subcutaneous injection, tail veininjection, spleen implantation, intraperitoneal implantation,implantation under the renal capsule, or orthopin implantation, e.g.,colon cancer cells implanted in colonic tissue. See, e.g., PCTpublication No. WO 97/33551, published Sep. 18, 1997. Probably the mostoften used animal species in oncological studies are immunodeficientmice and, in particular, nude mice. The observation that the nude mousewith thymic hypo/aplasia could successfully act as a host for humantumor xenografts has lead to its widespread use for this purpose. Theautosomal recessive nu gene has been introduced into a very large numberof distinct congenic strains of nude mouse, including, for example, ASW,A/He, AKR, BALB/c, B10. LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st,NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII, and SJL. In addition, awide variety of other animals with inherited immunological defects otherthan the nude mouse have been bred and used as recipients of tumorxenografts. For further details see, e.g., The Nude Mouse in OncologyResearch, E. Boven and B. Winograd, eds. (CRC Press, Inc., 1991).

The cells introduced into such animals can be derived from knowntumor/cancer cell lines, such as any of the above-listed tumor celllines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cellline transfected with the neu protooncogene); ras-transfected NIH-3T3cells; Caco-2 (ATCC HTB-37); or a moderately well-differentiated gradeII human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38); or fromtumors and cancers. Samples of tumor or cancer cells can be obtainedfrom patients undergoing surgery, using standard conditions involvingfreezing and storing in liquid nitrogen. Karmali et al., Br. J. Cancer,48: 689–696 (1983).

Tumor cells can be introduced into animals such as nude mice by avariety of procedures. The subcutaneous (s.c.) space in mice is verysuitable for tumor implantation. Tumors can be transplanted s.c. assolid blocks, as needle biopsies by use of a trochar, or as cellsuspensions. For solid-block or trochar implantation, tumor tissuefragments of suitable size are introduced into the s.c. space. Cellsuspensions are freshly prepared from primary tumors or stable tumorcell lines, and injected subcutaneously. Tumor cells can also beinjected as subdermal implants. In this location, the inoculum isdeposited between the lower part of the dermal connective tissue and thes.c. tissue.

Animal models of breast cancer can be generated, for example, byimplanting rat neuroblastoma cells (from which the neu oncogene wasinitially isolated), or neu-transformed NIH-3T3 cells into nude mice,essentially as described by Drebin et al. Proc. Nat. Acad. Sci. USA, 83:9129–9133 (1986).

Similarly, animal models of colon cancer can be generated by passagingcolon cancer cells in animals, e.g., nude mice, leading to theappearance of tumors in these animals. An orthotopic transplant model ofhuman colon cancer in nude mice has been described, for example, by Wanget al., Cancer Research, 54: 4726–4728 (1994) and Too et al., CancerResearch, 55: 681–684 (1995). This model is based on the so-called“METAMOUSE™” sold by AntiCancer, Inc., (San Diego, Calif.).

Tumors that arise in animals can be removed and cultured in vitro. Cellsfrom the in vitro cultures can then be passaged to animals. Such tumorscan serve as targets for further testing or drug screening.Alternatively, the tumors resulting from the passage can be isolated andRNA from pre-passage cells and cells isolated after one or more roundsof passage analyzed for differential expression of genes of interest.Such passaging techniques can be performed with any known tumor orcancer cell lines.

For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemicallyinduced fibrosarcomas of BALB/c female mice (DeLeo et al., J. Exp. Med.,146: 720 (1977)), which provide a highly controllable model system forstudying the anti-tumor activities of various agents. Palladino et al.,J. Immunol., 138: 4023–4032 (1987). Briefly, tumor cells are propagatedin vitro in cell culture. Prior to injection into the animals, the celllines are washed and suspended in buffer, at a cell density of about10×10⁶ to 10×10⁷ cells/ml. The animals are then infected subcutaneouslywith 10 to 100 μl of the cell suspension, allowing one to three weeksfor a tumor to appear.

In addition, the Lewis lung (3LL) carcinoma of mice, which is one of themost thoroughly studied experimental tumors, can be used as aninvestigational tumor model. Efficacy in this tumor model has beencorrelated with beneficial effects in the treatment of human patientsdiagnosed with small-cell carcinoma of the lung (SCCL). This tumor canbe introduced in normal mice upon injection of tumor fragments from anaffected mouse or of cells maintained in culture. Zupi et al., Br. J.Cancer, 41: suppl. 4, 30 (1980). Evidence indicates that tumors can bestarted from injection of even a single cell and that a very highproportion of infected tumor cells survive. For further informationabout this tumor model see, Zacharski, Haemostasis, 16: 300–320 (1986).

One way of evaluating the efficacy of a test compound in an animal modelwith an implanted tumor is to measure the size of the tumor before andafter treatment. Traditionally, the size of implanted tumors has beenmeasured with a slide caliper in two or three dimensions. The measurelimited to two dimensions does not accurately reflect the size of thetumor; therefore, it is usually converted into the corresponding volumeby using a mathematical formula. However, the measurement of tumor sizeis very inaccurate. The therapeutic effects of a drug candidate can bebetter described as treatment-induced growth delay and specific growthdelay. Another important variable in the description of tumor growth isthe tumor volume doubling time. Computer programs for the calculationand description of tumor growth are also available, such as the programreported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop onImmune-Deficient Animals, Wu and Sheng eds. (Basel, 1989), p. 301. It isnoted, however, that necrosis and inflammatory responses followingtreatment may actually result in an increase in tumor size, at leastinitially. Therefore, these changes need to be carefully monitored, by acombination of a morphometric method and flow cytometric analysis.

Further, recombinant (transgenic) animal models can be engineered byintroducing the coding portion of the PRO gene identified herein intothe genome of animals of interest, using standard techniques forproducing transgenic animals. Animals that can serve as a target fortransgenic manipulation include, without limitation, mice, rats,rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g.,baboons, chimpanzees and monkeys. Techniques known in the art tointroduce a transgene into such animals include pronucleicmicroinjection (U.S. Pat. No. 4,873,191); retrovirus-mediated genetransfer into germ lines (e.g., Van der Putten et al., Proc. Natl. Acad.Sci. USA, 82: 6148–615 (1985)); gene targeting in embryonic stem cells(Thompson et al., Cell, 56: 313–321 (1989)); electroporation of embryos(Lo, Mol. Cell. Biol., 3: 1803–1814 (1983)); and sperm-mediated genetransfer. Lavitrano et al., Cell, 57: 717–73 (1989). For a review, seefor example, U.S. Pat. No. 4,736,866.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA, 89: 6232–636 (1992). The expression of the transgene intransgenic animals can be monitored by standard techniques. For example,Southern blot analysis or PCR amplification can be used to verify theintegration of the transgene. The level of mRNA expression can then beanalyzed using techniques such as in situ hybridization, Northern blotanalysis, PCR, or immunocytochemistry. The animals are further examinedfor signs of tumor or cancer development.

Alternatively, “knock-out” animals can be constructed that have adefective or altered gene encoding a PRO polypeptide identified herein,as a result of homologous recombination between the endogenous geneencoding the PRO polypeptide and altered genomic DNA encoding the samepolypeptide introduced into an embryonic cell of the animal. Forexample, cDNA encoding a particular PRO polypeptide can be used to clonegenomic DNA encoding that polypeptide in accordance with establishedtechniques. A portion of the genomic DNA encoding a particular PROpolypeptide can be deleted or replaced with another gene, such as a geneencoding a selectable marker that can be used to monitor integration.Typically, several kilobases of unaltered flanking DNA (both at the 5′and 3′ ends) are included in the vector. See, e.g., Thomas and Capecchi,Cell, 51: 503 (1987) for a description of homologous recombinationvectors. The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected. See, e.g.,Li et al., Cell, 69: 915 (1992). The selected cells are then injectedinto a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras. See, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL:Oxford, 1987), pp. 113–152. A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm to create a “knock-out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized, for instance, by their ability to defend against certainpathological conditions and by their development of pathologicalconditions due to absence of the PRO polypeptide.

The efficacy of antibodies specifically binding the PRO polypeptidesidentified herein, and other drug candidates, can be tested also in thetreatment of spontaneous animal tumors. A suitable target for suchstudies is the feline oral squamous cell carcinoma (SCC). Feline oralSCC is a highly invasive, malignant tumor that is the most common oralmalignancy of cats, accounting for over 60% of the oral tumors reportedin this species. It rarely metastasizes to distant sites, although thislow incidence of metastasis may merely be a reflection of the shortsurvival times for cats with this tumor. These tumors are usually notamenable to surgery, primarily because of the anatomy of the feline oralcavity. At present, there is no effective treatment for this tumor.Prior to entry into the study, each cat undergoes complete clinicalexamination and biopsy, and is scanned by computed tomography (CT). Catsdiagnosed with sublingual oral squamous cell tumors are excluded fromthe study. The tongue can become paralyzed as a result of such tumor,and even if the treatment kills the tumor, the animals may not be ableto feed themselves. Each cat is treated repeatedly, over a longer periodof time. Photographs of the tumors will be taken daily during thetreatment period, and at each subsequent recheck. After treatment, eachcat undergoes another CT scan. CT scans and thoracic radiograms areevaluated every 8 weeks thereafter. The data are evaluated fordifferences in survival, response, and toxicity as compared to controlgroups. Positive response may require evidence of tumor regression,preferably with improvement of quality of life and/or increased lifespan.

In addition, other spontaneous animal tumors, such as fibrosarcoma,adenocarcinoma, lymphoma, chondroma, or leiomyosarcoma of dogs, cats,and baboons can also be tested. Of these, mammary adenocarcinoma in dogsand cats is a preferred model as its appearance and behavior are verysimilar to those in humans. However, the use of this model is limited bythe rare occurrence of this type of tumor in animals.

Other in vitro and in vivo cardiovascular, endothelial, and angiogenictests known in the art are also suitable herein.

5.2.4.2. Tissue Distribution

The results of the cardiovascular, endothelial, and angiogenic assaysherein can be verified by further studies, such as by determining mRNAexpression in various human tissues.

As noted before, gene amplification and/or gene expression in varioustissues may be measured by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201–5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.

Gene expression in various tissues, alternatively, may be measured byimmunological methods, such as immunohistochemical staining of tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native-sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope. General techniques forgenerating antibodies, and special protocols for in situ hybridizationare provided hereinbelow.

5.2.4.3. Antibody Binding Studies

The results of the cardiovascular, endothelial, and angiogenic study canbe further verified by antibody binding studies, in which the ability ofanti-PRO antibodies to inhibit the effect of the PRO polypeptides onendothelial cells or other cells used in the cardiovascular,endothelial, and angiogenic assays is tested. Exemplary antibodiesinclude polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies, the preparation of which will be describedhereinbelow.

Antibody binding studies may be carried out in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual ofTechniques (CRC Press, Inc., 1987), pp. 147–158.

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of target protein in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte that remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody that is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tissue sample may be fresh or frozen ormay be embedded in paraffin and fixed with a preservative such asformalin, for example.

5.2.4.4. Cell-Based Tumor Assays

Cell-based assays and animal models for cardiovascular, endothelial, andangiogenic disorders, such as tumors, can be used to verify the findingsof a cardiovascular, endothelial, and angiogenic assay herein, andfurther to understand the relationship between the genes identifiedherein and the development and pathogenesis of undesirablecardiovascular, endothelial, and angiogenic cell growth. The role ofgene products identified herein in the development and pathology ofundesirable cardiovascular, endothelial, and angiogenic cell growth,e.g., tumor cells, can be tested by using cells or cells lines that havebeen identified as being stimulated or inhibited by the PRO polypeptideherein. Such cells include, for example, those set forth in the Examplesbelow.

In a different approach, cells of a cell type known to be involved in aparticular cardiovascular, endothelial, and angiogenic disorder aretransfected with the cDNAs herein, and the ability of these cDNAs toinduce excessive growth or inhibit growth is analyzed. If thecardiovascular, endothelial, and angiogenic disorder is cancer, suitabletumor cells include, for example, stable tumor cell lines such as theB104-1-1 cell line (stable NIH-3T3 cell line transfected with the neuprotooncogene) and ras-transfected NIH-3T3 cells, which can betransfected with the desired gene and monitored for tumorigenic growth.Such transfected cell lines can then be used to test the ability ofpoly- or monoclonal antibodies or antibody compositions to inhibittumorigenic cell growth by exerting cytostatic or cytotoxic activity onthe growth of the transformed cells, or by mediating antibody-dependentcellular cytotoxicity (ADCC). Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of cardiovascular, endothelial, andangiogenic disorders such as cancer.

In addition, primary cultures derived from tumors in transgenic animals(as described above) can be used in the cell-based assays herein,although stable cell lines are preferred. Techniques to derivecontinuous cell lines from transgenic animals are well known in the art.See, e.g., Small et al., Mol. Cell. Biol., 5: 642–648 (1985).

5.2.4.5. Gene Therapy

Described below are methods and compositions whereby disease symptomsmay be ameliorated. Certain diseases are brought about, at least inpart, by an excessive level of gene product, or by the presence of agene product exhibiting an abnormal or excessive activity. As such, thereduction in the level and/or activity of such gene products would bringabout the amelioration of such disease symptoms.

Alternatively, certain other diseases are brought about, at least inpart, by the absence or reduction of the level of gene expression, or areduction in the level of a gene product's activity. As such, anincrease in the level of gene expression and/or the activity of suchgene products would bring about the amelioration of such diseasesymptoms.

In some cases, the up-regulation of a gene in a disease state reflects aprotective role for that gene product in responding to the diseasecondition. Enhancement of such a target gene's expression, or theactivity of the target gene product, will reinforce the protectiveeffect it exerts. Some disease states may result from an abnormally lowlevel of activity of such a protective gene. In these cases also, anincrease in the level of gene expression and/or the activity of suchgene products would bring about the amelioration of such diseasesymptoms.

The PRO polypeptides described herein and polypeptidyl agonists andantagonists may be employed in accordance with the present invention byexpression of such polypeptides in vivo, which is often referred to asgene therapy.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells: in vivo and ex vivo.For in vivo delivery the nucleic acid is injected directly into thepatient, usually at the sites where the PRO polypeptide is required,i.e., the site of synthesis of the PRO polypeptide, if known, and thesite (e.g., wound) where biological activity of the PRO polypeptide isneeded. For ex vivo treatment, the patient's cells are removed, thenucleic acid is introduced into these isolated cells, and the modifiedcells are administered to the patient either directly or, for example,encapsulated within porous membranes that are implanted into the patient(see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There area varietyof techniques available for introducing nucleic acids into viable cells.The techniques vary depending upon whether the nucleic acid istransferred into cultured cells in vitro, or transferred in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, transduction, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc.Transduction involves the association of a replication-defective,recombinant viral (preferably retroviral) particle with a cellularreceptor, followed by introduction of the nucleic acids contained by theparticle into the cell. A commonly used vector for ex vivo delivery ofthe gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral vectors (such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) andlipid-based systems (useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol; see, e.g., Tonkinson etal., Cancer Investigation, 14(1): 54–65 (1996)). The most preferredvectors for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral vector such asa retroviral vector includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger. Inaddition, a viral vector such as a retroviral vector includes a nucleicacid molecule that, when transcribed in the presence of a gene encodingthe PRO polypeptide, is operably linked thereto and acts as atranslation initiation sequence. Such vector constructs also include apackaging signal, long terminal repeats (LTRs) or portions thereof, andpositive and negative strand primer binding sites appropriate to thevirus used (if these are not already present in the viral vector). Inaddition, such vector typically includes a signal sequence for secretionof the PRO polypeptide from a host cell in which it is placed.Preferably the signal sequence for this purpose is a mammalian signalsequence, most preferably the native signal sequence for the PROpolypeptide. Optionally, the vector construct may also include a signalthat directs polyadenylation, as well as one or more restriction sitesand a translation termination sequence. By way of example, such vectorswill typically include a 5′ LTR, a tRNA binding site, a packagingsignal, an origin of second-strand DNA synthesis, and a 3′ LTR or aportion thereof. Other vectors can be used that are non-viral, such ascationic lipids, polylysine, and dendrimers.

In some situations, it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell-surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins that bind to a cell-surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins that undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262: 4429–4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87: 3410–3414 (1990). For a review of the currentlyknown gene marking and gene therapy protocols, see, Anderson et al.,Science, 256: 808–813 (1992). See also WO 93/25673 and the referencescited therein.

Suitable gene therapy and methods for making retroviral particles andstructural proteins can be found in, e.g., U.S. Pat. No. 5,681,746.

5.2.4.6. Use of Gene as a Diagnostic

This invention is also related to the use of the gene encoding the PROpolypeptide as a diagnostic. Detection of a mutated form of the PROpolypeptide will allow a diagnosis of a cardiovascular, endothelial, andangiogenic disease or a susceptibility to a cardiovascular, endothelial,and angiogenic disease, such as a tumor, since mutations in the PROpolypeptide may cause tumors.

Individuals carrying mutations in the genes encoding a human PROpolypeptide may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells, suchas from blood, urine, saliva, tissue biopsy, and autopsy material. Thegenomic DNA may be used directly for detection or maybe amplifiedenzymatically by using PCR (Saiki et al., Nature,324: 163–166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding thePRO polypeptide can be used to identify and analyze the PRO polypeptidemutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled RNA encoding the PRO polypeptide, or alternatively,radiolabeled antisense DNA sequences encoding the PRO polypeptide.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamidine gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures. See, e.g., Myerset al., Science, 230: 1242 (1985).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method, for example, Cotton et al., Proc. Natl. Acad. Sci. USA,85: 4397–4401 (1985).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing, or the use of restriction enzymes, e.g.,restriction fragment length polymorphisms (RFLP), and Southern blottingof genomic DNA.

5.2.4.7. Use to Detect PRO Polypeptide Levels

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

Expression of nucleic acid encoding the PRO polypeptide may be linked tovascular disease or neovascularization associated with tumor formation.If the PRO polypeptide has a signal sequence and the mRNA is highlyexpressed in endothelial cells and to a lesser extent in smooth musclecells, this indicates that the PRO polypeptide is present in serum.Accordingly, an anti-PRO polypeptide antibody could be used to diagnosevascular disease or neovascularization associated with tumor formation,since an altered level of this PRO polypeptide may be indicative of suchdisorders.

A competition assay may be employed wherein antibodies specific to thePRO polypeptide are attached to a solid support and the labeled PROpolypeptide and a sample derived from the host are passed over the solidsupport and the amount of label detected attached to the solid supportcan be correlated to a quantity of the PRO polypeptide in the sample.

5.2.4.8. Chromosome Mapping

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15–25 bp) from the cDNA. Computer analysis for the3′-untranslated region is used to rapidly select primers that do notspan more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes, andpreselection by hybridization to construct chromosome-specific cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 500 or 600bases; however, clones larger than 2,000 bp have a higher likelihood ofbinding to a unique chromosomal location with sufficient signalintensity for simple detection. FISH requires use of the clones fromwhich the gene encoding the PRO polypeptide was derived, and the longerthe better. For example, 2,000 bp is good, 4,000 bp is better, and morethan 4,000 is probably not necessary to get good results a reasonablepercentage of the time. For a review of this technique, see, Verma etal., Human Chromosomes: a Manual of Basic Techniques (Pergamon Press,New York, 1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available online through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region is thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

5.2.4.9. Screening Assays for Drug Candidates

This invention encompasses methods of screening compounds to identifythose that mimic the PRO polypeptide (agonists) or prevent the effect ofthe PRO polypeptide (antagonists). Screening assays for antagonist drugcandidates are designed to identify compounds that bind or complex withthe PRO polypeptide encoded by the genes identified herein, or otherwiseinterfere with the interaction of the encoded polypeptides with othercellular proteins. Such screening assays will include assays amenable tohigh-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO polypeptide encoded by the gene identified herein orthe drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the PRO polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the PROpolypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340: 245–246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578–9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789–5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a PROpolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

If the PRO polypeptide has the ability to stimulate the proliferation ofendothelial cells in the presence of the co-mitogen ConA, then oneexample of a screening method takes advantage of this ability.Specifically, in the proliferation assay, human umbilical veinendothelial cells are obtained and cultured in 96-well flat-bottomedculture plates (Costar, Cambridge, Mass.) and supplemented with areaction mixture appropriate for facilitating proliferation of thecells, the mixture containing Con-A (Calbiochem, La Jolla, Calif.).Con-A and the compound to be screened are added and after incubation at37° C., cultures are pulsed with ³-H-thymidine and harvested onto glassfiber filters (phD; Cambridge Technology, Watertown, Mass.). Mean³-H-thymidine incorporation (cpm) of triplicate cultures is determinedusing a liquid scintillation counter (Beckman Instruments, Irvine,Calif.). Significant ³-(H)-thymidine incorporation indicates stimulationof endothelial cell proliferation.

To assay for antagonists, the assay described above is performed;however, in this assay the PRO polypeptide is added along with thecompound to be screened and the ability of the compound to inhibit³-(H)thymidine incorporation in the presence of the PRO polypeptideindicates that the compound is an antagonist to the PRO polypeptide.Alternatively, antagonists may be detected by combining the PROpolypeptide and a potential antagonist with membrane-bound PROpolypeptide receptors or recombinant receptors under appropriateconditions for a competitive inhibition assay. The PRO polypeptide canbe labeled, such as by radioactivity, such that the number of PROpolypeptide molecules bound to the receptor can be used to determine theeffectiveness of the potential antagonist. The gene encoding thereceptor can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting. Coligan etal., Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the PRO polypeptide and a cDNA library createdfrom this RNA is divided into pools and used to transfect COS cells orother cells that are not responsive to the PRO polypeptide. Transfectedcells that are grown on glass slides are exposed to the labeled PROpolypeptide. The PRO polypeptide can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to autoradiographic analysis. Positive pools areidentified and sub-pools are prepared and re-transfected using aninteractive sub-pooling and re-screening process, eventually yielding asingle clone that encodes the putative receptor.

As an alternative approach for receptor identification, the labeled PROpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with the labeledPRO polypeptide in the presence of the candidate compound. The abilityof the compound to enhance or block this interaction could then bemeasured.

The compositions useful in the treatment of cardiovascular, endothelial,and angiogenic disorders include, without limitation, antibodies, smallorganic and inorganic molecules, peptides, phosphopeptides, antisenseand ribozyme molecules, triple-helix molecules, etc., that inhibit theexpression and/or activity of the target gene product.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with a PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

Another potential PRO polypeptide antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see, Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. A sequence “complementary” to aportion of an RNA, as referred to herein, means a sequence havingsufficient complementarity to be able to hybridize with the RNA, forminga stable duplex; in the case of double-stranded antisense nucleic acids,a single strand of the duplex DNA may thus be tested, or triplex helixformation may be assayed. The ability to hybridize will depend on boththe degree of complementarity and the length of the antisense nucleicacid. Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the PRO polypeptide (antisense—Okano, Neurochem.,56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression (CRC Press: Boca Raton, Fla., 1988).

The antisense oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger, et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553–6556; Lemaitre, et al., 1987, Proc. Natl. Acad. Sci.U.S.A. 84:648–652; PCT Publication No. WO88/09810, published Dec. 15,1988) or the blood-brain barrier (see, e.g., PCT Publication No.WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavageagents (see, e.g., Krol et al., 1988, BioTechniques 6:958–976) orintercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539–549). Tothis end, the oligonucleotide may be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier, et al.,1987, Nucl. Acids Res. 15:6625–6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue, et al., 1987, Nucl. Acids Res.15:6131–6148), or a chimeric RNA-DNA analogue (Inoue, et al., 1987, FEBSLett. 215:327–330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein, et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin, et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448–7451), etc.

The oligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the PRO polypeptide. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation-initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Antisense RNA or DNA molecules are generally at least about 5 bases inlength, about 10 bases in length, about 15 bases in length, about 20bases in length, about 25 bases in length, about 30 bases in length,about 35 bases in length, about 40 bases in length, about 45 bases inlength, about 50 bases in length, about 55 bases in length, about 60bases in length, about 65 bases in length, about 70 bases in length,about 75 bases in length, about 80 bases in length, about 85 bases inlength, about 90 bases in length, about 95 bases in length, about 100bases in length, or more.

Potential antagonists further include small molecules that bind to theactive site, the receptor binding site, or growth factor or otherrelevant binding site of the PRO polypeptide, thereby blocking thenormal biological activity of the PRO polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

Additional potential antagonists are ribozymes, which are enzymatic RNAmolecules capable of catalyzing the specific cleavage of RNA. Ribozymesact by sequence-specific hybridization to the complementary target RNA,followed by endonucleolytic cleavage. Specific ribozyme cleavage siteswithin a potential RNA target can be identified by known techniques. Forfurther details see, e.g., Rossi, Current Biology, 4: 469–471 (1994),and PCT publication No. WO 97/33551 (published Sep. 18, 1997).

While ribozymes that cleave mRNA at site specific recognition sequencescan be used to destroy target gene mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions which form complementary base pairs withthe target mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Myers, 1995, Molecular Biology andBiotechnology: A Comprehensive Desk Reference, VCH Publishers, New York,(see especially FIG. 4, page 833) and in Haseloff and Gerlach, 1988,Nature, 334:585–591, which is incorporated herein by reference in itsentirety.

Preferably the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the target gene mRNA, i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574–578; Zaug andCech, 1986, Science, 231:470–475; Zaug, et al., 1986, Nature,324:429–433; published International patent application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207–216). TheCech-type ribozymes have an eight base pair active site that hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes that targeteight base-pair active site sequences that are present in the targetgene.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells that express the target gene in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous target gene messagesand inhibit translation. Because ribozymes, unlike antisense molecules,are catalytic, a lower intracellular concentration is required forefficiency.

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

5.2.4.10. Types of Cardiovascular, Endothelial, and Angiogenic Disordersto be Treated

The PRO polypeptides, or agonists or antagonists thereto, that haveactivity in the cardiovascular, angiogenic, and endothelial assaysdescribed herein, and/or whose gene product has been found to belocalized to the cardiovascular system, are likely to have therapeuticuses in a variety of cardiovascular, endothelial, and angiogenicdisorders, including systemic disorders that affect vessels, such asdiabetes mellitus. Their therapeutic utility could include diseases ofthe arteries, capillaries, veins, and/or lymphatics. Examples oftreatments hereunder include treating muscle wasting disease, treatingosteoporosis, aiding in implant fixation to stimulate the growth ofcells around the implant and therefore facilitate its attachment to itsintended site, increasing IGF stability in tissues or in serum, ifapplicable, and increasing binding to the IGF receptor (since IGF hasbeen shown in vitro to enhance human marrow erythroid and granulocyticprogenitor cell growth).

The PRO polypeptides or agonists or antagonists thereto may also beemployed to stimulate erythropoiesis or granulopoiesis, to stimulatewound healing or tissue regeneration and associated therapies concernedwith re-growth of tissue, such as connective tissue, skin, bone,cartilage, muscle, lung, or kidney, to promote angiogenesis, tostimulate or inhibit migration of endothelial cells, and to proliferatethe growth of vascular smooth muscle and endothelial cell production.The increase in angiogenesis mediated by the PRO polypeptide or agonistwould be beneficial to ischemic tissues and to collateral coronarydevelopment in the heart subsequent to coronary stenosis. Antagonistsare used to inhibit the action of such polypeptides, for example, tolimit the production of excess connective tissue during wound healing orpulmonary fibrosis if the PRO polypeptide promotes such production. Thiswould include treatment of acute myocardial infarction and heartfailure.

Moreover, the present invention provides the treatment of cardiachypertrophy, regardless of the underlying cause, by administering atherapeutically effective dose of the PRO polypeptide, or agonist orantagonist thereto. If the objective is the treatment of human patients,the PRO polypeptide preferably is recombinant human PRO polypeptide(rhPRO polypeptide). The treatment for cardiac hypertrophy can beperformed at any of its various stages, which may result from a varietyof diverse pathologic conditions, including myocardial infarction,hypertension, hypertrophic cardiomyopathy, and valvular regurgitation.The treatment extends to all stages of the progression of cardiachypertrophy, with or without structural damage of the heart muscle,regardless of the underlying cardiac disorder.

The decision of whether to use the molecule itself or an agonist thereoffor any particular indication, as opposed to an antagonist to themolecule, would depend mainly on whether the molecule herein promotescardiovascularization, genesis of endothelial cells, or angiogenesis orinhibits these conditions. For example, if the molecule promotesangiogenesis, an antagonist thereof would be useful for treatment ofdisorders where it is desired to limit or prevent angiogenesis. Examplesof such disorders include vascular tumors such as haemangioma, tumorangiogenesis, neovascularization in the retina, choroid, or cornea,associated with diabetic retinopathy or premature infant retinopathy ormacular degeneration and proliferative vitreoretinopathy, rheumatoidarthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation,psoriasis, endometriosis associated with neovascularization, restenosissubsequent to balloon angioplasty, scar tissue overproduction, forexample, that seen in a keloid that forms after surgery, fibrosis aftermyocardial infarction, or fibrotic lesions associated with pulmonaryfibrosis.

If, however, the molecule inhibits angiogenesis, it would be expected tobe used directly for treatment of the above conditions.

On the other hand, if the molecule stimulates angiogenesis it would beused itself (or an agonist thereof) for indications where angiogenesisis desired such as peripheral vascular disease, hypertension,inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon,aneurysms, arterial restenosis, thrombophlebitis, lymphangitis,lymphedema, wound healing and tissue repair, ischemia reperfusioninjury, angina, myocardial infarctions such as acute myocardialinfarctions, chronic heart conditions, heart failure such as congestiveheart failure, and osteoporosis.

If, however, the molecule inhibits angiogenesis, an antagonist thereofwould be used for treatment of those conditions where angiogenesis isdesired.

Specific types of diseases are described below, where the PROpolypeptide herein or agonists or antagonists thereof may serve asuseful for vascular-related drug targeting or as therapeutic targets forthe treatment or prevention of the disorders. Atherosclerosis is adisease characterized by accumulation of plaques of intimal thickeningin arteries, due to accumulation of lipids, proliferation of smoothmuscle cells, and formation of fibrous tissue within the arterial wall.The disease can affect large, medium, and small arteries in any organ.Changes in endothelial and vascular smooth muscle cell function areknown to play an important role in modulating the accumulation andregression of these plaques.

Hypertension is characterized by raised vascular pressure in thesystemic arterial, pulmonary arterial, or portal venous systems.Elevated pressure may result from or result in impaired endothelialfunction and/or vascular disease.

Inflammatory vasculitides include giant cell arteritis, Takayasu'sarteritis, polyarteritis nodosa (including the microangiopathic form),Kawasaki's disease, microscopic polyangiitis, Wegener's granulomatosis,and a variety of infectious-related vascular disorders (includingHenoch-Schonlein prupura). Altered endothelial cell function has beenshown to be important in these diseases.

Reynaud's disease and Reynaud's phenomenon are characterized byintermittent abnormal impairment of the circulation through theextremities on exposure to cold. Altered endothelial cell function hasbeen shown to be important in this disease.

Aneurysms are saccular or fusiform dilatations of the arterial or venoustree that are associated with altered endothelial cell and/or vascularsmooth muscle cells.

Arterial restenosis (restenosis of the arterial wall) may occurfollowing angioplasty as a result of alteration in the function andproliferation of endothelial and vascular smooth muscle cells.

Thrombophlebitis and lymphangitis are inflammatory disorders of veinsand lymphatics, respectively, that may result from, and/or in, alteredendothelial cell function. Similarly, lymphedema is a conditioninvolving impaired lymphatic vessels resulting from endothelial cellfunction.

The family of benign and malignant vascular tumors are characterized byabnormal proliferation and growth of cellular elements of the vascularsystem. For example, lymphangiomas are benign tumors of the lymphaticsystem that are congenital, often cystic, malformations of thelymphatics that usually occur in newborns. Cystic tumors tend to growinto the adjacent tissue. Cystic tumors usually occur in the cervicaland axillary region. They can also occur in the soft tissue of theextremities. The main symptoms are dilated, sometimes reticular,structured lymphatics and lymphocysts surrounded by connective tissue.Lymphangiomas are assumed to be caused by improperly connected embryoniclymphatics or their deficiency. The result is impaired local lymphdrainage. Griener et al., Lymphology, 4: 140–144 (1971).

Another use for the PRO polypeptides herein or agonists or antagoniststhereto is in the prevention of tumor angiogenesis, which involvesvascularization of a tumor to enable it to growth and/or metastasize.This process is dependent on the growth of new blood vessels. Examplesof neoplasms and related conditions that involve tumor angiogenesisinclude breast carcinomas, lung carcinomas, gastric carcinomas,esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovariancarcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endometrialcarcinoma, endometrial hyperplasia, endometriosis, fibrosarcomas,choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma,laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skincarcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreascarcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroidcarcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma,abnormal vascular proliferation associated with phakomatoses, edema(such as that associated with brain tumors), and Meigs' syndrome.

Age-related macular degeneration (AMD) is a leading cause of severevisual loss in the elderly population. The exudative form of AMD ischaracterized by choroidal neovascularization and retinal pigmentepithelial cell detachment. Because choroidal neovascularization isassociated with a dramatic worsening in prognosis, the PRO polypeptideor agonist or antagonist thereto is expected to be useful in reducingthe severity of AMD.

Healing of trauma such as wound healing and tissue repair is also atargeted use for the PRO polypeptides herein or their agonists orantagonists. Formation and regression of new blood vessels is essentialfor tissue healing and repair. This category includes bone, cartilage,tendon, ligament, and/or nerve tissue growth or regeneration, as well aswound healing and tissue repair and replacement, and in the treatment ofburns, incisions, and ulcers. A PRO polypeptide or agonist or antagonistthereof that induces cartilage and/or bone growth in circumstances wherebone is not normally formed has application in the healing of bonefractures and cartilage damage or defects in humans and other animals.Such a preparation employing a PRO polypeptide or agonist or antagonistthereof may have prophylactic use in closed as well as open fracturereduction and also in the improved fixation of artificial joints. Denovo bone formation induced by an osteogenic agent contributes to therepair of congenital, trauma-induced, or oncologic, resection-inducedcraniofacial defects, and also is useful in cosmetic plastic surgery.

PRO polypeptides or agonists or antagonists thereto may also be usefulto promote better or faster closure of non-healing wounds, includingwithout limitation pressure ulcers, ulcers associated with vascularinsufficiency, surgical and traumatic wounds, and the like.

It is expected that a PRO polypeptide or agonist or antagonist theretomay also exhibit activity for generation or regeneration of othertissues, such as organs (including, for example, pancreas, liver,intestine, kidney, skin, or endothelium), muscle (smooth, skeletal, orcardiac), and vascular (including vascular endothelium) tissue, or forpromoting the growth of cells comprising such tissues. Part of thedesired effects may be by inhibition or modulation of fibrotic scarringto allow normal tissue to regenerate.

A PRO polypeptide herein or agonist or antagonist thereto may also beuseful for gut protection or regeneration and treatment of lung or liverfibrosis, reperfusion injury in various tissues, and conditionsresulting from systemic cytokine damage. Also, the PRO polypeptide oragonist or antagonist thereto may be useful for promoting or inhibitingdifferentiation of tissues described above from precursor tissues orcells, or for inhibiting the growth of tissues described above.

A PRO polypeptide or agonist or antagonist thereto may also be used inthe treatment of periodontal diseases and in other tooth-repairprocesses. Such agents may provide an environment to attractbone-forming cells, stimulate growth of bone-forming cells, or inducedifferentiation of progenitors of bone-forming cells. A PRO polypeptideherein or an agonist or an antagonist thereto may also be useful in thetreatment of osteoporosis or osteoarthritis, such as through stimulationof bone and/or cartilage repair or by blocking inflammation or processesof tissue destruction (collagenase activity, osteoclast activity, etc.)mediated by inflammatory processes, since blood vessels play animportant role in the regulation of bone turnover and growth.

Another category of tissue regeneration activity that may beattributable to the PRO polypeptide herein or agonist or antagonistthereto is tendon/ligament formation. A protein that inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed has application in the healingof tendon or ligament tears, deformities, and other tendon or ligamentdefects in humans and other animals. Such a preparation may haveprophylactic use in preventing damage to tendon or ligament tissue, aswell as use in the improved fixation of tendon or ligament to bone orother tissues, and in repairing defects to tendon or ligament tissue. Denovo tendon/ligament-like tissue formation induced by a composition ofthe PRO polypeptide herein or agonist or antagonist thereto contributesto the repair of congenital, trauma-induced, or other tendon or ligamentdefects of other origin, and is also useful in cosmetic plastic surgeryfor attachment or repair of tendons or ligaments. The compositionsherein may provide an environment to attract tendon- or ligament-formingcells, stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions herein may also beuseful in the treatment of tendinitis, carpal tunnel syndrome, and othertendon or ligament defects. The compositions may also include anappropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

The PRO polypeptide or its agonist or antagonist may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e., for the treatment of central and peripheral nervous systemdisease and neuropathies, as well as mechanical and traumatic disorders,that involve degeneration, death, or trauma to neural cells or nervetissue. More specifically, a PRO polypeptide or its agonist orantagonist may be used in the treatment of diseases of the peripheralnervous system, such as peripheral nerve injuries, peripheral neuropathyand localized neuropathies, and central nervous system diseases, such asAlzheimer's, Parkinson's disease, Huntington's disease, amyotrophiclateral sclerosis, and Shy-Drager syndrome. Further conditions that maybe treated in accordance with the present invention include mechanicaland traumatic disorders, such as spinal cord disorders, head trauma, andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a PRO polypeptide herein or agonist or antagonistthereto.

Ischemia-reperfusion injury is another indication. Endothelial celldysfunction may be important in both the initiation of, and inregulation of the sequelae of events that occur followingischemia-reperfusion injury.

Rheumatoid arthritis is a further indication. Blood vessel growth andtargeting of inflammatory cells through the vasculature is an importantcomponent in the pathogenesis of rheumatoid and sero-negative forms ofarthritis.

A PRO polypeptide or its agonist or antagonist may also be administeredprophylactically to patients with cardiac hypertrophy, to prevent theprogression of the condition, and avoid sudden death, including death ofasymptomatic patients. Such preventative therapy is particularlywarranted in the case of patients diagnosed with massive leftventricular cardiac hypertrophy (a maximal wall thickness of 35 mm ormore in adults, or a comparable value in children), or in instances whenthe hemodynamic burden on the heart is particularly strong.

A PRO polypeptide or its agonist or antagonist may also be useful in themanagement of atrial fibrillation, which develops in a substantialportion of patients diagnosed with hypertrophic cardiomyopathy.

Further indications include angina, myocardial infarctions such as acutemyocardial infarctions, and heart failure such as congestive heartfailure. Additional non-neoplastic conditions include psoriasis,diabetic and other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, thyroidhyperplasias (including Grave's disease), corneal and other tissuetransplantation, chronic inflammation, lung inflammation, nephroticsyndrome, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

In view of the above, the PRO polypeptides or agonists or antagoniststhereof described herein, which are shown to alter or impact endothelialcell function, proliferation, and/or form, are likely to play animportant role in the etiology and pathogenesis of many or all of thedisorders noted above, and as such can serve as therapeutic targets toaugment or inhibit these processes or for vascular-related drugtargeting in these disorders.

5.2.4.11. Administration Protocols, Schedules, Doses, and Formulations

The molecules herein and agonists and antagonists thereto arepharmaceutically useful as a prophylactic and therapeutic agent forvarious disorders and diseases as set forth above.

Therapeutic compositions of the PRO polypeptides or agonists orantagonists are prepared for storage by mixing the desired moleculehaving the appropriate degree of purity with optional pharmaceuticallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Additional examples of such carriers include ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts, or electrolytes such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, and polyethylene glycol.Carriers for topical or gel-based forms of agonist or antagonist includepolysaccharides such as sodium carboxymethylcellulose ormethylcellulose, polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols. For all administrations, conventional depot formsare suitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations. The PROpolypeptides or agonists or antagonists will typically be formulated insuch vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml.

Another formulation comprises incorporating a PRO polypeptide or agonistor antagonist thereof into formed articles. Such articles can be used inmodulating endothelial cell growth and angiogenesis. In addition, tumorinvasion and metastasis may be modulated with these articles.

PRO polypeptides or agonists or antagonists to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. PRO polypeptides ordinarily will bestored in lyophilized form or in solution if administered systemically.If in lyophilized form, the PRO polypeptide or agonist or antagonistthereto is typically formulated in combination with other ingredientsfor reconstitution with an appropriate diluent at the time for use. Anexample of a liquid formulation of a PRO polypeptide or agonist orantagonist is a sterile, clear, colorless unpreserved solution filled ina single-dose vial for subcutaneous injection. Preserved pharmaceuticalcompositions suitable for repeated use may contain, for example,depending mainly on the indication and type of polypeptide:

-   -   a) PRO polypeptide or agonist or antagonist thereto;    -   b) a buffer capable of maintaining the pH in a range of maximum        stability of the polypeptide or other molecule in solution,        preferably about 4–8;    -   c) a detergent/surfactant primarily to stabilize the polypeptide        or molecule against agitation-induced aggregation;    -   d) an isotonifier;    -   e) a preservative selected from the group of phenol, benzyl        alcohol and a benzethonium halide, e.g., chloride; and    -   f) water.

If the detergent employed is non-ionic, it may, for example, bepolysorbates (e.g., POLYSORBATE™ (TWEEN™) 20, 80, etc.) or poloxamers(e.g., POLOXAMER™ 188). The use of non-ionic surfactants permits theformulation to be exposed to shear surface stresses without causingdenaturation of the polypeptide. Further, such surfactant-containingformulations may be employed in aerosol devices such as those used in apulmonary dosing, and needleless jet injector guns (see, e.g., EP257,956).

An isotonifier may be present to ensure isotonicity of a liquidcomposition of the PRO polypeptide or agonist or antagonist thereto, andincludes polyhydric sugar alcohols, preferably trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, andmannitol. These sugar alcohols can be used alone or in combination.Alternatively, sodium chloride or other appropriate inorganic salts maybe used to render the solutions isotonic.

The buffer may, for example, be an acetate, citrate, succinate, orphosphate buffer depending on the pH desired. The pH of one type ofliquid formulation of this invention is buffered in the range of about 4to 8, preferably about physiological pH.

The preservatives phenol, benzyl alcohol and benzethonium halides, e.g.,chloride, are known antimicrobial agents that may be employed.

Therapeutic PRO polypeptide compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle. The formulations are preferably administered asrepeated intravenous (i.v.), subcutaneous (s.c.), or intramuscular(i.m.) injections, or as aerosol formulations suitable for intranasal orintrapulmonary delivery (for intrapulmonary delivery see, e.g., EP257,956).

PRO polypeptides can also be administered in the form ofsustained-released preparations. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the protein, which matrices are in the form ofshaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (e.g.,poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J.Biomed. Mater. Res., 15: 167–277 (1981) and Langer, Chem. Tech., 12:98–105 (1982) or poly(vinylalcohol)), polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547–556 (1983)),non-degradable ethylene-vinyl acetate (Langer et al., supra), degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid (EP133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release PRO polypeptide compositions also include liposomallyentrapped PRO polypeptides. Liposomes containing the PRO polypeptide areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688–3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. USA, 77: 4030–4034 (1980); EP 52,322; EP 36,676; EP 88,046;EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomesare of the small (about 200–800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal therapy.

The therapeutically effective dose of a PRO polypeptide or agonist orantagonist thereto will, of course, vary depending on such factors asthe pathological condition to be treated (including prevention), themethod of administration, the type of compound being used for treatment,any co-therapy involved, the patient's age, weight, general medicalcondition, medical history, etc., and its determination is well withinthe skill of a practicing physician. Accordingly, it will be necessaryfor the therapist to titer the dosage and modify the route ofadministration as required to obtain the maximal therapeutic effect. Ifthe PRO polypeptide has a narrow host range, for the treatment of humanpatients formulations comprising human PRO polypeptide, more preferablynative-sequence human PRO polypeptide, are preferred. The clinician willadminister the PRO polypeptide until a dosage is reached that achievesthe desired effect for treatment of the condition in question. Forexample, if the objective is the treatment of CHF, the amount would beone that inhibits the progressive cardiac hypertrophy associated withthis condition. The progress of this therapy is easily monitored by echocardiography. Similarly, in patients with hypertrophic cardiomyopathy,the PRO polypeptide can be administered on an empirical basis.

With the above guidelines, the effective dose generally is within therange of from about 0.001 to about 1.0 mg/kg, more preferably about0.01–1.0 mg/kg, most preferably about 0.01–0.1 mg/kg.

For non-oral use in treating human adult hypertension, it isadvantageous to administer the PRO polypeptide in the form of aninjection at about 0.01 to 50 mg, preferably about 0.05 to 20 mg, mostpreferably 1 to 20 mg, per kg body weight, 1 to 3 times daily byintravenous injection. For oral administration, a molecule based on thePRO polypeptide is preferably administered at about 5 mg to 1 g,preferably about 10 to 100 mg, per kg body weight, 1 to 3 times daily.It should be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less than 0.5 ng/mg protein.Moreover, for human administration, the formulations preferably meetsterility, pyrogenicity, general safety, and purity as required by FDAOffice and Biologics standards.

The dosage regimen of a pharmaceutical composition containing the PROpolypeptide to be used in tissue regeneration will be determined by theattending physician considering various factors that modify the actionof the polypeptides, e.g., amount of tissue weight desired to be formed,the site of damage, the condition of the damaged tissue, the size of awound, type of damaged tissue (e.g., bone), the patient's age, sex, anddiet, the severity of any infection, time of administration, and otherclinical factors. The dosage may vary with the type of matrix used inthe reconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF-I, to the final composition may also affectthe dosage. Progress can be monitored by periodic assessment oftissue/bone growth and/or repair, for example, X-rays, histomorphometricdeterminations, and tetracycline labeling.

The route of PRO polypeptide or antagonist or agonist administration isin accord with known methods, e.g., by injection or infusion byintravenous, intramuscular, intracerebral, intraperitoneal,intracerobrospinal, subcutaneous, intraocular, intraarticular,intrasynovial, intrathecal, oral, topical, or inhalation routes, or bysustained-release systems as noted below. The PRO polypeptide or agonistor antagonists thereof also are suitably administered by intratumoral,peritumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. The intraperitoneal route isexpected to be particularly useful, for example, in the treatment ofovarian tumors.

If a peptide or small molecule is employed as an antagonist or agonist,it is preferably administered orally or non-orally in the form of aliquid or solid to mammals.

Examples of pharmacologically acceptable salts of molecules that formsalts and are useful hereunder include alkali metal salts (e.g., sodiumsalt, potassium salt), alkaline earth metal salts (e.g., calcium salt,magnesium salt), ammonium salts, organic base salts (e.g., pyridinesalt, triethylamine salt), inorganic acid salts (e.g., hydrochloride,sulfate, nitrate), and salts of organic acid (e.g., acetate, oxalate,p-toluenesulfonate).

For compositions herein that are useful for bone, cartilage, tendon, orligament regeneration, the therapeutic method includes administering thecomposition topically, systemically, or locally as an implant or device.When administered, the therapeutic composition for use is in apyrogen-free, physiologically acceptable form. Further, the compositionmay desirably be encapsulated or injected in a viscous form for deliveryto the site of bone, cartilage, or tissue damage. Topical administrationmay be suitable for wound healing and tissue repair. Preferably, forbone and/or cartilage formation, the composition would include a matrixcapable of delivering the protein-containing composition to the site ofbone and/or cartilage damage, providing a structure for the developingbone and cartilage and preferably capable of being resorbed into thebody. Such matrices may be formed of materials presently in use forother implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance, andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid,polyglycolic acid, and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above-mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability.

One specific embodiment is a 50:50 (mole weight) copolymer of lacticacid and glycolic acid in the form of porous particles having diametersranging from 150 to 800 microns. In some applications, it will be usefulto utilize a sequestering agent, such as carboxymethyl cellulose orautologous blood clot, to prevent the polypeptide compositions fromdisassociating from the matrix.

One suitable family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydoxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, andcarboxymethylcellulose, one preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer, and poly(vinyl alcohol).The amount of sequestering agent useful herein is 0.5–20 wt %,preferably 1–10 wt %, based on total formulation weight, whichrepresents the amount necessary to prevent desorption of the polypeptide(or its antagonist) from the polymer matrix and to provide appropriatehandling of the composition, yet not so much that the progenitor cellsare prevented from infiltrating the matrix, thereby providing thepolypeptide (or its antagonist) the opportunity to assist the osteogenicactivity of the progenitor cells.

5.2.4.12. Combination Therapies

The effectiveness of the PRO polypeptide or an agonist or antagonistthereof in preventing or treating the disorder in question may beimproved by administering the active agent serially or in combinationwith another agent that is effective for those purposes, either in thesame composition or as separate compositions.

For example, for treatment of cardiac hypertrophy, PRO polypeptidetherapy can be combined with the administration of inhibitors of knowncardiac myocyte hypertrophy factors, e.g., inhibitors of α-adrenergicagonists such as phenylephrine; endothelin-1 inhibitors such asBOSENTAN™ and MOXONODIN™; inhibitors to CT-1 (U.S. Pat. No. 5,679,545);inhibitors to LIF; ACE inhibitors; des-aspartate-angiotensin Iinhibitors (U.S. Pat. No. 5,773,415), and angiotensin II inhibitors.

For treatment of cardiac hypertrophy associated with hypertension, thePRO polypeptide can be administered in combination with β-adrenergicreceptor blocking agents, e.g., propranolol, timolol, tertalolol,carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol,metoprolol, or carvedilol; ACE inhibitors, e.g., quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, or lisinopril; diuretics,e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, orindapamide; and/or calcium channel blockers, e g., diltiazem,nifedipine, verapamil, or nicardipine. Pharmaceutical compositionscomprising the therapeutic agents identified herein by their genericnames are commercially available, and are to be administered followingthe manufacturers' instructions for dosage, administration, adverseeffects, contraindications, etc. See, e.g., Physicians' Desk Reference(Medical Economics Data Production Co.: Montvale, N.J., 1997), 51thEdition.

Preferred candidates for combination therapy in the treatment ofhypertrophic cardiomyopathy are β-adrenergic-blocking drugs (e.g.,propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol,penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol),verapamil, difedipine, or diltiazem. Treatment of hypertrophy associatedwith high blood pressure may require the use of antihypertensive drugtherapy, using calcium channel blockers, e.g., diltiazem, nifedipine,verapamil, or nicardipine; β-adrenergic blocking agents; diuretics,e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, orindapamide; and/or ACE-inhibitors, e.g., quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, or lisinopril.

For other indications, PRO polypeptides or their agonists or antagonistsmay be combined with other agents beneficial to the treatment of thebone and/or cartilage defect, wound, or tissue in question. These agentsinclude various growth factors such as EGF, PDGF, TGF-α or TGF-β, IGF,FGF, and CTGF.

In addition, PRO polypeptides or their agonists or antagonists used totreat cancer may be combined with cytotoxic, chemotherapeutic, orgrowth-inhibitory agents as identified above. Also, for cancertreatment, the PRO polypeptide or agonist or antagonist thereof issuitably administered serially or in combination with radiologicaltreatments, whether involving irradiation or administration ofradioactive substances.

The effective amounts of the therapeutic agents administered incombination with the PRO polypeptide or agonist or antagonist thereofwill be at the physician's or veterinarian's discretion. Dosageadministration and adjustment is done to achieve maximal management ofthe conditions to be treated. For example, for treating hypertension,these amounts ideally take into account use of diuretics or digitalis,and conditions such as hyper- or hypotension, renal impairment, etc. Thedose will additionally depend on such factors as the type of thetherapeutic agent to be used and the specific patient being treated.Typically, the amount employed will be the same dose as that used, ifthe given therapeutic agent is administered without the PRO polypeptide.

5.2.4.13. Articles of Manufacture

An article of manufacture such as a kit containing the PRO polypeptideor agonists or antagonists thereof useful for the diagnosis or treatmentof the disorders described above comprises at least a container and alabel. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionthat is effective for diagnosing or treating the condition and may havea sterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agent in the composition is the PROpolypeptide or an agonist or antagonist thereto. The label on, orassociated with, the container indicates that the composition is usedfor diagnosing or treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use. The article of manufacture may also comprisea second or third container with another active agent as describedabove.

5.2.5. Antibodies

Some of the most promising drug candidates according to the presentinvention are antibodies and antibody fragments that may inhibit theproduction or the gene product of the genes identified herein and/orreduce the activity of the gene products.

5.2.5.1. Polyclonal Antibodies

Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the PRO polypeptide or afusion protein thereof. It may be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Examples of such immunogenic proteins include, but are notlimited to, keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants thatmay be employed include Freund's complete adjuvant and MPL-TDM adjuvant(monophosphoryl Lipid A or synthetic trehalose dicorynomycolate). Theimmunization protocol may be selected by one skilled in the art withoutundue experimentation.

5.2.5.2. Monoclonal Antibodies

The anti-PRO antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the PRO polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, Monoclonal Antibodies: Principles and Practice (New York:Academic Press, 1986), pp. 59–103. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine, and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high-level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications (MarcelDekker, Inc.: New York, 1987) pp. 51–63.

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thePRO polypeptide. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, supra. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy- and light-chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison et al, supra) or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fe region so as to prevent heavy-chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart.

5.2.5.3. Human and Humanized Antibodies

The anti-PRO antibodies may further comprise humanized antibodies orhuman antibodies. Humanized forms of non-human (e.g., murine) antibodiesare chimeric immunoglobulins, immunoglobulin chains, or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)₂, or other antigen-bindingsubsequences of antibodies) that contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residues thatare found neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin, and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody preferably also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Jones et al., Nature, 321: 522–525 (1986); Riechmann etal., Nature, 332: 323–329 (1988); Presta, Curr. Op. Struct. Biol.,2:593–596 (1992).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321: 522–525 (1986); Riechmann et al., Nature,332: 323–327(1988); Verhoeyen et al., Science, 239: 1534–1536 (1988)),by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries. Hoogenboom and Winter, J.Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581(1991). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies. Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1): 86–95 (1991). Similarly,human antibodies can be made by introducing human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed that closely resemblesthat seen in humans in all respects, including gene rearrangement,assembly, and antibody repertoire. This approach is described, forexample, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016, and in the following scientific publications:Marks et al., Bio/Technology, 10: 779–783 (1992); Lonberg et al.,Nature, 368: 856–859 (1994); Morrison, Nature, 368: 812–813 (1994);Fishwild et al., Nature Biotechnology, 14: 845–851 (1996); Neuberger,Nature Biotechnology, 14: 826 (1996); Lonberg and Huszar, Intern. Rev.Immunol., 13: 65–93 (1995).

5.2.5.4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO polypeptide, the other one is for any other antigen, andpreferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities. Milsteinand Cuello, Nature, 305: 537–539 (1983). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655–3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant-domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies, see, for example,Suresh et al., Methods in Enzymology, 121: 210 (1986).

5.2.5.5. Heteroconjugate Antibodies

Heteroconjugate antibodies are composed of two covalently joinedantibodies. Such antibodies have, for example, been proposed to targetimmune-system cells to unwanted cells (U.S. Pat. No. 4,676,980), and fortreatment of HIV infection. WO 91/00360; WO 92/200373; EP 03089. It iscontemplated that the antibodies may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

5.2.5.6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See, Caron et al., J. Exp. Med., 176: 1191–1195(1992) and Shopes, J. Immunol., 148: 2918–2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., CancerResearch, 53: 2560–2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See, Stevenson et al.,Anti-Cancer Drug Design, 3: 219–230 (1989).

5.2.5.7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

5.2.5.8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286–288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See, Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

5.2.5.9. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersas noted above and below in the form of pharmaceutical compositions.

If the PRO polypeptide is intracellular and whole antibodies are used asinhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889–7893 (1993).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

5.2.5.10. Methods of Treatment Using the Antibody

It is contemplated that the antibodies to a PRO polypeptide may be usedto treat various cardiovascular, endothelial, and angiogenic conditionsas noted above.

The antibodies are administered to a mammal, preferably a human, inaccord with known methods, such as intravenous administration as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous administration of the antibody is preferred.

Other therapeutic regimens may be combined with the administration ofthe antibodies of the instant invention as noted above. For example, ifthe antibodies are to treat cancer, the patient to be treated with suchantibodies may also receive radiation therapy. Alternatively, or inaddition, a chemotherapeutic agent may be administered to the patient.Preparation and dosing schedules for such chemotherapeutic agents may beused according to manufacturers' instructions or as determinedempirically by the skilled practitioner. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService, Ed., M. C. Perry (Williams & Wilkins: Baltimore, Md., 1992).The chemotherapeutic agent may precede, or follow administration of theantibody, or may be given simultaneously therewith. The antibody may becombined with an anti-estrogen compound such as tamoxifen or EVISTA™ oran anti-progesterone such as onapristone (see, EP 616812) in dosagesknown for such molecules.

If the antibodies are used for treating cancer, it may be desirable alsoto administer antibodies against other tumor-associated antigens, suchas antibodies that bind to one or more of the ErbB2, EGFR, ErbB3, ErbB4,or VEGF receptor(s). These also include the agents set forth above.Also, the antibody is suitably administered serially or in combinationwith radiological treatments, whether involving irradiation oradministration of radioactive substances. Alternatively, or in addition,two or more antibodies binding the same or two or more differentantigens disclosed herein may be co-administered to the patient.Sometimes, it may be beneficial also to administer one or more cytokinesto the patient. In a preferred embodiment, the antibodies herein areco-administered with a growth-inhibitory agent. For example, thegrowth-inhibitory agent may be administered first, followed by anantibody of the present invention. However, simultaneous administrationor administration of the antibody of the present invention first is alsocontemplated. Suitable dosages for the growth-inhibitory agent are thosepresently used and may be lowered due to the combined action (synergy)of the growth-inhibitory agent and the antibody herein.

In one embodiment, vascularization of tumors is attacked in combinationtherapy. The anti-PRO polypeptide antibody and another antibody (e.g.,anti-VEGF) are administered to tumor-bearing patients at therapeuticallyeffective doses as determined, for example, by observing necrosis of thetumor or its metastatic foci, if any. This therapy is continued untilsuch time as no further beneficial effect is observed or clinicalexamination shows no trace of the tumor or any metastatic foci. Then TNFis administered, alone or in combination with an auxiliary agent such asalpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin,anti-heregulin antibody, D-factor, interleukin-1 (IL-1), interleukin-2(IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), oragents that promote microvascular coagulation in tumors, such asanti-protein C antibody, anti-protein S antibody, or C4b binding protein(see, WO 91/01753, published Feb. 21, 1991), or heat or radiation.

Since the auxiliary agents will vary in their effectiveness, it isdesirable to compare their impact on the tumor by matrix screening inconventional fashion. The administration of anti-PRO polypeptideantibody and TNF is repeated until the desired clinical effect isachieved. Alternatively, the anti-PRO polypeptide antibody isadministered together with TNF and, optionally, auxiliary agent(s). Ininstances where solid tumors are found in the limbs or in otherlocations susceptible to isolation from the general circulation, thetherapeutic agents described herein are administered to the isolatedtumor or organ. In other embodiments, a FGF or PDGF antagonist, such asan anti-FGF or an anti-PDGF neutralizing antibody, is administered tothe patient in conjunction with the anti-PRO polypeptide antibody.Treatment with anti-PRO polypeptide antibodies preferably may besuspended during periods of wound healing or desirableneovascularization.

For the prevention or treatment of cardiovascular, endothelial, andangiogenic disorder, the appropriate dosage of an antibody herein willdepend on the type of disorder to be treated, as defined above, theseverity and course of the disease, whether the antibody is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments.

For example, depending on the type and severity of the disorder, about 1μg/kg to 50 mg/kg (e.g., 0.1–20 mg/kg) of antibody is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily or weekly dosage might range from about 1μg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is repeated or sustained until a desiredsuppression of disorder symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays, including, for example, radiographictumor imaging.

5.2.5.11. Articles of Manufacture with Antibodies

An article of manufacture containing a container with the antibody and alabel is also provided. Such articles are described above, wherein theactive agent is an anti-PRO antibody.

5.2.5.12. Diagnosis and Prognosis of Tumors Using Antibodies

If the indication for which the antibodies are used is cancer, whilecell-surface proteins, such as growth receptors over expressed incertain tumors, are excellent targets for drug candidates or tumor(e.g., cancer) treatment, the same proteins along with PRO polypeptidesfind additional use in the diagnosis and prognosis of tumors. Forexample, antibodies directed against the PRO polypeptides may be used astumor diagnostics or prognostics.

For example, antibodies, including antibody fragments, can be usedqualitatively or quantitatively to detect the expression of genesincluding the gene encoding the PRO polypeptide. The antibody preferablyis equipped with a detectable, e.g., fluorescent label, and binding canbe monitored by light microscopy, flow cytometry, fluorimetry, or othertechniques known in the art. Such binding assays are performedessentially as described above.

In situ detection of antibody binding to the marker gene products can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent to those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

The following Examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

The disclosures of all patent and literature references cited in thepresent specification are hereby incorporated by reference in theirentirety.

6. EXAMPLES

Commercially available reagents referred to in the Examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following Examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., supra; Ausubel et al., Current Protocols inMolecular Biology (Green Publishing Associates and Wiley Interscience,N.Y., 1989); Innis et al., PCR Protocols: A Guide to Methods andApplications (Academic Press, Inc.: N.Y., 1990); Harlow et al.,Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold SpringHarbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford,1984); Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

6.1. Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ®, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology, 266:460–480 (1996)) asa comparison of the ECD protein sequences to a 6 frame translation ofthe EST sequences. Those comparisons with a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

Using this extracellular domain homology screen, consensus DNA sequenceswere assembled relative to the other identified EST sequences usingphrap. In addition, the consensus DNA sequences obtained were often (butnot always) extended using repeated cycles of BLAST or BLAST-2 and phrapto extend the consensus sequence as far as possible using the sources ofEST sequences discussed above.

Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100–1000 bp in length. The probe sequences are typically 40–55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1 –1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed bystandard methods using commercially available reagents such as thosefrom Invitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain theSfiI site; see, Holmes et al., Science, 253:1278–1280 (1991)) in theunique XhoI and NotI sites.

6.2. Example 2 Isolation of cDNA Clones by Amylase Screening

6.2.1. Preparation of Oligo dT Primed cDNA Library

mRNA was isolated from a human tissue of interest using reagents andprotocols from Invitrogen, San Diego, Calif. (Fast Track 2). This RNAwas used to generate an oligo dT primed cDNA library in the vector pRK5Dusing reagents and protocols from Life Technologies, Gaithersburg, Md.(Super Script Plasmid System). In this procedure, the double strandedcDNA was sized to greater than 1000 bp and the SalI/NotI linkered cDNAwas cloned into XhoI/NotI cleaved vector. pRK5D is a cloning vector thathas an sp6 transcription initiation site followed by an SfiI restrictionenzyme site preceding the XhoI/NotI cDNA cloning sites.

6.2.2. Preparation of Random Primed cDNA Library

A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500–1000 bp, linkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

6.2.3. Transformation and Detection

DNA from the library described in paragraph 2 above was chilled on Iceto which was added electrocompetent DH10B bacteria (Life Technologies,20 ml). The bacteria and vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (LifeTechnologies, 1 ml) was added and the mixture was incubated at 37° C.for 30 minutes. The transformants were then plated onto 20 standard 150mm LB plates containing ampicillin and incubated for 16 hours (37° C.).Positive colonies were scraped off the plates and the DNA was isolatedfrom the bacterial pellet using standard protocols, e.g., CsCl-gradient.The purified DNA was then carried on to the yeast protocols below.

The yeast methods were divided into three categories: (1) Transformationof yeast with the plasmid/cDNA combined vector; (2) Detection andisolation of yeast clones secreting amylase; and (3) PCR amplificationof the insert directly from the yeast colony and purification of the DNAfor sequencing and further analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain has thefollowing genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

Transformation was performed based on the protocol outlined by Gietz etal., Nucl. Acid. Res., 20:1425 (1992). Transformed cells were theninoculated from agar into YEPD complex media broth (100 ml) and grownovernight at 30° C. The YEPD broth was prepared as described in Kaiseret al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., p. 207 (1994). The overnight culture was then diluted toabout 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPD broth (500 ml)and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀=0.4–0.5).

The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

Transformation took place by mixing the prepared cells (100 μl) withfreshly denatured single stranded salmon testes DNA (Lofstrand Labs,Gaithersburg, Md.) and transforming DNA (1 μg, vol.<10 μl) in microfugetubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mMLi₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5–10 seconds, decanted andresuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

Alternatively, instead of multiple small reactions, the transformationwas performed using a single, large scale reaction, wherein reagentamounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lackinguracil (SCD-Ura) prepared as described in Kaiser et al., Methods inYeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p.208–210 (1994). Transformants were grown at 30° C. for 2–3 days.

The detection of colonies secreting amylase was performed by includingred starch in the selective growth media. Starch was coupled to the reddye (Reactive Red-120, Sigma) as per the procedure described by Biely etal., Anal. Biochem, 172:176–179 (1988). The coupled starch wasincorporated into the SCD-Ura agar plates at a final concentration of0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50–100 mM final concentration).

The positive colonies were picked and streaked across fresh selectivemedia (onto 150 mm plates) in order to obtain well isolated andidentifiable single colonies. Well isolated single colonies positive foramylase secretion were detected by direct incorporation of red starchinto buffered SCD-Ura agar. Positive colonies were determined by theirability to break down starch resulting in a clear halo around thepositive colony visualized directly.

6.2.4. Isolation of DNA by PCR Amplification

When a positive colony was isolated, a portion of it was picked by atoothpick and diluted into sterile water (30 μl) in a 96 well plate. Atthis time, the positive colonies were either frozen and stored forsubsequent analysis or immediately amplified. An aliquot of cells (5 μl)was used as a template for the PCR reaction m a 25 μl volume containing:0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mM dNTP's(Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μl forwardoligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water.

The sequence of the forward oligonucleotide 1 was: (SEQ ID NO:382)5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′ The sequence ofreverse oligonucleotide 2 was: (SEQ ID NO:383)5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′

PCR was then performed as follows: a. Denature 92° C.,  5 minutes b.  3cycles of: Denature 92° C., 30 seconds Anneal 59° C., 30 seconds Extend72° C., 60 seconds c.  3 cycles of: Denature 92° C., 30 seconds Anneal57° C., 30 seconds Extend 72° C., 60 seconds d. 25 cycles of: Denature92° C., 30 seconds Anneal 55° C., 30 seconds Extend 72° C., 60 secondse. Hold  4° C.

The underlined regions of the oligonucleotides annealed to the ADHpromoter region and the amylase region, respectively, and amplified a307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

Following the PCR, an aliquot of the reaction (5 μl) was examined byagarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA(TBE) buffering system as described by Sambrook et al., supra. Clonesresulting in a single strong PCR product larger than 400 bp were furtheranalyzed by DNA sequencing after purification with a 96 Qiaquick PCRclean-up column (Qiagen Inc., Chatsworth, Calif.).

6.3. Example 3 Isolation of cDNA Clones Using Signal Algorithm Analysis

Various polypeptide-encoding nucleic acid sequences were identified byapplying a proprietary signal sequence finding algorithm developed byGenentech, Inc., (South San Francisco, Calif.) upon ESTs as well asclustered and assembled EST fragments from public (e.g., GenBank) and/orprivate (LIFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, Calif.)databases. The signal sequence algorithm computes a secretion signalscore based on the character of the DNA nucleotides surrounding thefirst and optionally the second methionine codon(s) (ATG) at the 5′-endof the sequence or sequence fragment under consideration. Thenucleotides following the first ATG must code for at least 35unambiguous amino acids without any stop codons. If the first ATG hasthe required amino acids, the second is not examined. If neither meetsthe requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals. Use of thisalgorithm resulted in the identification of numerouspolypeptide-encoding nucleic acid sequences.

6.4. Example 4 Isolation of cDNA Clones Encoding Human PRO Polypeptides

Using the techniques described in Examples 1 to 3 above, numerousfull-length cDNA clones were identified as encoding PRO polypeptides asdisclosed herein. These cDNAs were then deposited under the terms of theBudapest Treaty with the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110–2209, USA (ATCC) as shown in Table7 below.

TABLE 7 Material ATCC Dep. No. Deposit Date 23330-1390 209775 Apr. 14,1998 23339-1130 209282 Sep. 18, 1997 26846-1397 203406 Oct. 27, 199826847-1395 209772 Apr. 14, 1998 27865-1091 209296 Sep. 23, 199730868-1156 1437-PTA Mar. 2, 2000 30871-1157 209380 Oct. 16, 199732286-1191 209385 Oct. 16, 1997 33089-1132 209262 Sep. 16, 199733092-1202 209420 Oct. 28, 1997 33100-1159 209377 Oct. 16, 199733223-1136 209264 Sep. 16, 1997 34392-1170 209526 Dec. 10, 199734431-1177 209399 Oct. 17, 1997 34433-1308 209719 Mar. 31, 199834434-1139 209252 Sep. 16, 1997 35600-1162 209370 Oct. 16, 199735673-1201 209418 Oct. 28, 1997 35880-1160 209379 Oct. 16, 199735918-1174 209402 Oct. 17, 1997 36350-1158 209378 Oct. 16, 199736638-1056 209456 Nov. 12, 1997 38268-1188 209421 Oct. 28, 199740370-1217 209485 Nov. 21, 1997 40628-1216 209432 Nov. 7, 199743316-1237 209487 Nov. 21, 1997 44196-1353 209847 May 6, 1998 45409-2511203579 Jan. 12, 1999 45419-1252 209616 Feb. 5, 1998 46777-1253 209619Feb. 5, 1998 48336-1309 209669 Mar. 11, 1998 48606-1479 203040 Jul. 1,1998 49435-1219 209480 Nov. 21, 1997 49631-1328 209806 Apr. 28, 199850919-1361 209848 May 6, 1998 50920-1325 209700 Mar. 26, 1998 50921-1458209859 May 12, 1998 52758-1399 209773 Apr. 14, 1998 53517-1366-1 209802Apr. 23, 1998 53915-1258 209593 Jan. 21, 1998 53974-1401 209774 Apr. 14,1998 53987-1438 209858 May 12, 1998 56047-1456 209948 Jun. 9, 199856050-1455 203011 Jun. 23, 1998 56110-1437 203113 Aug. 11, 199856405-1357 209849 May 6, 1998 56433-1406 209857 May 12, 1998 56439-1376209864 May 14, 1998 56529-1647 203293 Sep. 29, 1998 56865-1491 203022Jun. 23, 1998 56965-1356 209842 May 6, 1998 57033-1403-1 209905 May 27,1998 57037-1444 209903 May 27, 1998 57039-1402 209777 Apr. 14, 199857689-1385 209869 May 14, 1998 57690-1374 209950 Jun. 9, 1998 57694-1341203017 Jun. 23, 1998 57695-1340 203006 Jun. 23, 1998 57699-1412 203020Jun. 23, 1998 57700-1408 203583 Jan. 12, 1999 57708-1411 203021 Jun. 23,1998 57838-1337 203014 Jun. 23, 1998 58847-1383 209879 May 20, 199858852-1637 203271 Sep. 22, 1998 58853-1423 203016 Jun. 23, 199859212-1627 203245 Sep. 9, 1998 59220-1514 209962 Jun. 9, 1998 59493-1420203050 Jul. 1, 1998 59497-1496 209941 Jun. 4, 1998 59586-1520 203288Sep. 29, 1998 59588-1571 203106 Aug. 11, 1998 59620-1463 209989 Jun. 16,1998 59622-1334 209984 Jun. 16, 1998 59777-1480 203111 Aug. 11, 199859848-1512 203088 Aug. 4, 1998 59849-1504 209986 Jun. 16, 199860621-1516 203091 Aug. 4, 1998 60622-1525 203090 Aug. 4, 1998 60764-1533203452 Nov. 10, 1998 60783-1611 203130 Aug. 18, 1998 61755-1554 203112Aug. 11, 1998 62306-1570 203254 Sep. 9, 1998 62312-2558 203836 Mar. 9,1999 62814-1521 203093 Aug. 4, 1998 62872-1509 203100 Aug. 4, 199864883-1526 203253 Sep. 9, 1998 64886-1601 203241 Sep. 9, 1998 64889-1541203250 Sep. 9, 1998 64896-1539 203238 Sep. 9, 1998 64897-1628 203216Sep. 15, 1998 64903-1553 203223 Sep. 15, 1998 64908-1163-1 203243 Sep.9, 1998 64950-1590 203224 Sep. 15, 1998 65402-1540 203252 Sep. 9, 199865404-1551 203244 Sep. 9, 1998 65405-1547 203476 Nov. 17, 199865410-1569 203231 Sep. 15, 1998 65412-1523 203094 Aug. 4, 199866307-2661 431-PTA Jul. 27, 1999 66526-1616 203246 Sep. 9, 199866659-1593 203269 Sep. 22, 1998 66660-1585 203279 Sep. 22, 199866667-1596 203267 Sep. 22, 1998 66672-1586 203265 Sep. 22, 199866675-1587 203282 Sep. 22, 1998 67300-1605 203163 Aug. 25, 199868818-2536 203657 Feb. 9, 1999 68862-2546 203652 Feb. 9, 1999 68872-1620203160 Aug. 25, 1998 71290-1630 203275 Sep. 22, 1998 73736-1657 203466Nov. 17, 1998 73739-1645 203270 Sep. 22, 1998 73742-1662 203316 Oct. 6,1998 76385-1692 203664 Feb. 9, 1999 76393-1664 203323 Oct. 6, 199876399-1700 203472 Nov. 17, 1998 76400-2528 203573 Jan. 12, 199976510-2504 203477 Nov. 17, 1998 76529-1666 203315 Oct. 6, 199876532-1702 203473 Nov. 17, 1998 76541-1675 203409 Oct. 27, 199877503-1686 203362 Oct. 20, 1998 77624-2515 203553 Dec. 22, 199879230-2525 203549 Dec. 22, 1998 79862-2522 203550 Dec. 22, 199880145-2594 204-PTA Jun. 8, 1999 80899-2501 203539 Dec. 15, 199881754-2532 203542 Dec. 15, 1998 81757-2512 203543 Dec. 15, 199881761-2583 203862 Mar. 23, 1999 82358-2738 510-PTA Aug. 10, 199982364-2538 203603 Jan. 20, 1999 82403-2959 2317-PTA Aug. 1, 200083500-2506 203391 Oct. 29, 1998 83560-2569 203816 Mar. 2, 199984210-2576 203818 Mar. 2, 1999 84920-2614 203966 Apr. 27, 199986576-2595 203868 Mar. 23, 1999 92218-2554 203834 Mar. 9, 199992233-2599 134-PTA May 25, 1999 92256-2596 203891 Mar. 30, 199992265-2669 256-PTA Jun. 22, 1999 92274-2617 203971 Apr. 27, 199992929-2534-1 203586 Jan. 12, 1999 93011-2637 20-PTA May 4, 199994854-2586 203864 Mar. 23, 1999 96787-2534-1 203589 Jan. 12, 199996867-2620 203972 Apr. 27, 1999 96872-2674 550-PTA Aug. 17, 199996878-2626 23-PTA May 4, 1999 96889-2641 119-PTA May 25, 1999100312-2645 44-PTA May 11, 1999 105782-2693 387-PTA Jul. 20, 1999105849-2704 473-PTA Aug. 3, 1999 108725-2766 863-PTA Oct. 19, 1999108769-2765 861-PTA Oct. 19, 1999 119498-2965 2298-PTA Jul. 25, 2000119535-2756 613-PTA Aug. 31, 1999 125185-2806 1031-PTA Dec. 7, 1999131639-2874 1784-PTA Apr. 25, 2000 139623-2893 1670-PTA Apr. 11, 2000143076-2787 1028-PTA Dec. 7, 1999 143276-2975 2387-PTA Aug. 8, 2000164625-2890 1535-PTA Mar. 21, 2000 167678-2963 2302-PTA Jul. 25, 2000170021-2923 1906-PTA May 23, 2000 170212-3000 2583-PTA Oct. 10, 2000177313-2982 2251-PTA Jul. 19, 2000

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit and for at least five (5) years afterthe most recent request for the furnishing of a sample of the depositreceived by the depository. The deposits will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures that all restrictionsimposed by the depositor on the availability of the deposited materialwill be irrevocably removed upon the granting of the pertinent U.S.patent, assures permanent and unrestricted availability of the progenyof the culture of the deposit to the public upon issuance of thepertinent U.S. patent or upon laying open to the public of any U.S. orforeign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

6.5 Example 5 Isolation of cDNA Clones Encoding Human PRO1873, PRO7223,PRO7248, PRO730, PRO532, PRO7261, PRO734, PRO771, PRO2010, PRO5723,PRO3444, PRO9940, PRO3562, PRO10008, PRO5730, PRO6008, PRO4527, PRO4538and PRO4553

DNA molecules encoding the PRO1873, PRO7223, PRO7248, PRO730, PRO532,PRO7261, PRO734, PRO771, PRO2010, PRO5723, PRO3444, PRO9940, PRO3562,PRO10008, PRO5730, PRO6008, PRO4527, PRO4538 and PRO4553 polypeptidesshown in the accompanying figures were obtained through GenBank.

6.6. Example 6 Use of PRO as a Hybridization Probe

The following method describes use of a nucleotide sequence encoding PROas a hybridization probe.

DNA comprising the coding sequence of full-length or mature PRO (asshown in accompanying figures) or a fragment thereof is employed as aprobe to screen for homologous DNAs (such as those encodingnaturally-occurring variants of PRO) in human tissue cDNA libraries orhuman tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high-stringency conditions. Hybridizationof radiolabeled probe derived from the gene encoding PRO polypeptide tothe filters is performed in a solution of 50% formamide, 5×SSC, 0.1%SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours.Washing of the filters is performed in an aqueous solution of 0.1×SSCand 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence can then be identified using standardtechniques known in the art.

6.7. Example 7 Expression of PRO in E. coli

This example illustrates preparation of an unglycosylated form of PRO byrecombinant expression in E. coli.

The DNA sequence encoding PRO is initially amplified using selected PCRprimers. The primers should contain restriction enzyme sites whichcorrespond to the restriction enzyme sites on the selected expressionvector. A variety of expression vectors may be employed. An example of asuitable vector is pBR322 (derived from E. coli; see, Bolivar et al.,Gene, 2:95 (1977)) which contains genes for ampicillin and tetracyclineresistance. The vector is digested with restriction enzyme anddephosphorylated. The PCR amplified sequences are then ligated into thevector. The vector will preferably include sequences which encode for anantibiotic resistance gene, a trp promoter, a poly-His leader (includingthe first six STII codons, poly-His sequence, and enterokinase cleavagesite), the PRO coding region, lambda transcriptional terminator, and anargU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO may be expressed in E. coli in a poly-His tagged form, using thefollowing procedure. The DNA encoding PRO is initially amplified usingselected PCR primers. The primers will contain restriction enzyme siteswhich correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an OD₆₀₀ of 3–5 is reached. Cultures are thendiluted 50–100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate.2H₂O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20–30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6–10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3–5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi²⁺-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12–36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2–10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A₂₈₀ absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO polypeptide are pooled andthe acetonitrile removed using a gentle stream of nitrogen directed atthe solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14M sodium chloride and 4% mannitol by dialysis or by gel filtration usingG25 Superfine (Pharmacia) resins equilibrated in the formulation bufferand sterile filtered.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as descibed above.

6.8. Example 8 Expression of PRO in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof PRO by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO DNA is ligated into pRK5with selected restriction enzymes to allow insertion of the PRO DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-PRO.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-PRODNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappayaet al, Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl,0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added, dropwise, 500 μlof 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and a precipitateis allowed to form for 10 minutes at 25° C. The precipitate is suspendedand added to the 293 cells and allowed to settle for about four hours at37° C. The culture medium is aspirated off and 2 ml of 20% glycerol inPBS is added for 30 seconds. The 293 cells are then washed with serumfree medium, fresh medium is added and the cells are incubated for about5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of the PRO polypeptide. The cultures containing transfectedcells may undergo further incubation (in serum free medium) and themedium is tested in selected bioassays.

In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing expressed PRO can then be concentrated andpurified by any selected method, such as dialysis and/or columnchromatography.

In another embodiment, PRO can be expressed in CHO cells. The pRK5-PROcan be transfected into CHO cells using known reagents such as CaPO₄ orDEAE-dextran. As described above, the cell cultures can be incubated,and the medium replaced with culture medium (alone) or medium containinga radiolabel such as ³⁵S-methionine. After determining the presence of aPRO polypeptide, the culture medium may be replaced with serum freemedium. Preferably, the cultures are incubated for about 6 days, andthen the conditioned medium is harvested.

The medium containing the expressed PRO polypeptide can then beconcentrated and purified by any selected method.

Epitope-tagged PRO may also be expressed in host CHO cells. The PRO maybe subcloned out of the pRK5 vector. The subclone insert can undergo PCRto fuse in frame with a selected epitope tag such as a poly-His tag intoa Baculovirus expression vector. The poly-His tagged PRO insert can thenbe subcloned into a SV40 driven vector containing a selection markersuch as DHFR for selection of stable clones. Finally, the CHO cells canbe transfected (as described above) with the SV40 driven vector.Labeling may be performed, as described above, to verify expression. Theculture medium containing the expressed poly-His tagged PRO can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g., extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or as a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used in expression in CHOcells is as described in Lucas et al., Nucl. Acids Res., 24:9 (1774–1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Qiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement into awater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 ml of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 ml of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 ml spinner containing 90 ml of selective media. After 1–2days, the cells are transferred into a 250 ml spinner filled with 150 mlselective growth medium and incubated at 37° C. After another 2–3 days,250 ml, 500 ml and 2000 ml spinners are seeded with 3×10⁵ cells/ml. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3L production spinner isseeded at 1.2×10⁶ cells/ml. On day 0, the cell number and pH isdetermined. On day 1, the spinner is sampled and sparging with filteredair is commenced. On day 2, the spinner is sampled, the temperatureshifted to 33° C., and 30 ml of 500 g/L glucose and 0.6 ml of 10%antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365Medical Grade Emulsion) taken. Throughout the production, the pH isadjusted as necessary to keep it at around 7.2. After 10 days, or untilthe viability drops below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate iseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi²⁺-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni²⁺-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4–5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μl of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as descibed above.

6.9. Example 9 Expression of PRO in Yeast

The following method describes recombinant expression of PRO in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding PRO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO. For secretion, DNA encoding PRO can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO signal peptide or other mammalian signal peptide, or, for example, ayeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression of PRO.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO can subsequently be isolated and purified by removingthe yeast cells from the fermentation medium by centrifugation and thenconcentrating the medium using selected cartridge filters. Theconcentrate containing PRO may further be purified using selected columnchromatography resins.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

6.10. Example 10 Expression of PRO in Baculovirus-Infected Insect Cells

The following method describes recombinant expression inBaculovirus-infected insect cells.

The sequence coding for PRO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-His tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO or the desired portion of the coding sequence ofPRO (such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular) is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4–5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-His tagged PRO can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175–179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 ml, washed with 25 ml of water and equilibrated with 25ml of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 ml per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM imidazole gradient in the secondary wash buffer. One ml fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged PRO are pooled and dialyzedagainst loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO can beperformed using known chromatography techniques, including for instance,Protein A or protein G column chromatography.

Following PCR amplification, the respective coding sequences aresubcloned into a baculovirus expression vector (pb.PH.IgG for IgGfusions and pb.PH.His.c for poly-His tagged proteins), and the vectorand Baculogold® baculovirus DNA (Pharmingen) are co-transfected into 105Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711), using Lipofectin(Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of thecommercially available baculovirus expression vector pVL1393(Pharmingen), with modified polylinker regions to include the His or Fctag sequences. The cells are grown in Hink's TNM-FH medium supplementedwith 10% FBS (Hyclone). Cells are incubated for 5 days at 28° C. Thesupernatant is harvested and subsequently used for the first viralamplification by infecting Sf9 cells in Hink's TNM-FH mediumsupplemented with 10% FBS at an approximate multiplicity of infection(MOI) of 10. Cells are incubated for 3 days at 28° C. The supernatant isharvested and the expression of the constructs in the baculovirusexpression vector is determined by batch binding of 1 ml of supernatantto 25 ml of Ni²⁺-NTA beads (QIAGEN) for histidine tagged proteins orProtein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteinsfollowed by SDS-PAGE analysis comparing to a known concentration ofprotein standard by Coomassie blue staining.

The first viral amplification supernatant is used to infect a spinnerculture (500 ml) of Sf9 cells grown in ESF-921 medium (ExpressionSystems LLC) at an approximate MOI of 0.1. Cells are incubated for 3days at 28° C. The supernatant is harvested and filtered. Batch bindingand SDS-PAGE analysis is repeated, as necessary, until expression of thespinner culture is confirmed.

The conditioned medium from the transfected cells (0.5 to 3 L) isharvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteinconstruct is purified using a Ni²⁺-NTA column (Qiagen). Beforepurification, imidazole is added to the conditioned media to aconcentration of 5 mM. The conditioned media is pumped onto a 6 mlNi²⁺-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing0.3 M NaCl and 5 mM imidazole at a flow rate of 4–5 ml/min. at 4° C.After loading, the column is washed with additional equilibration bufferand the protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein is subsequently desalted into astorage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at −80° C.

Immunoadhesin (Fc containing) constructs of proteins are purified fromthe conditioned media as follows. The conditioned media is pumped onto a5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mMNa phosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 ml of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity of the proteins is verified by SDS polyacrylamide gel (PEG)electrophoresis and N-terminal amino acid sequencing by Edmandegradation.

Alternatively, a modified baculovirus procedure may be usedincorporating high-5 cells. In this procedure, the DNA encoding thedesired sequence is amplified with suitable systems, such as Pfu(Stratagene), or fused upstream (5′-of) of an epitope tag contained witha baculovirus expression vector. Such epitope tags include poly-His tagsand immunoglobulin tags (like Fc regions of IgG). A variety of plasmidsmay be employed, including plasmids derived from commercially availableplasmids such as pIE1-1 (Novagen). The pIE1-1 and pIE1-2 vectors aredesigned for constitutive expression of recombinant proteins from thebaculovirus ie1 promoter in stably-transformed insect cells (1). Theplasmids differ only in the orientation of the multiple cloning sitesand contain all promoter sequences known to be important forie1-mediated gene expression in uninfected insect cells as well as thehr5 enhancer element. pIE1-1 and pIE1-2 include the translationinitiation site and can be used to produce fusion proteins. Briefly, thedesired sequence or the desired portion of the sequence (such as thesequence encoding the extracellular domain of a transmembrane protein)is amplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product is then digested with those selected restrictionenzymes and subcloned into the expression vector. For example,derivatives of pIE1-1 can include the Fc region of human IgG (pb.PH.IgG)or an 8 histidine (pb.PH.His) tag downstream (3′-of) the desiredsequence. Preferably, the vector construct is sequenced forconfirmation.

High-5 cells are grown to a con fluency of 50% under the conditions of,27° C., no CO₂, NO pen/strep. For each 150 mm plate, 30 μg of pIE basedvector containing the sequence is mixed with 1 ml Ex-Cell medium (Media:Ex-Cell 401+1/100 L-Glu JRH Biosciences #14401-78P (note: this media islight sensitive)), and in a separate tube, 100 μl of CellFectin(CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) is mixed with 1 mlof Ex-Cell medium. The two solutions are combined and allowed toincubate at room temperature for 15 minutes. 8 ml of Ex-Cell media isadded to the 2 ml of DNA/CellFECTIN mix and this is layered on high-5cells that have been washed once with Ex-Cell media. The plate is thenincubated in darkness for 1 hour at room temperature. The DNA/CellFECTINmix is then aspirated, and the cells are washed once with Ex-Cell toremove excess CellFECTIN, 30 ml of fresh Ex-Cell media is added and thecells are incubated for 3 days at 28° C. The supernatant is harvestedand the expression of the sequence in the baculovirus expression vectoris determined by batch binding of 1 ml of supernatent to 25 ml ofNi²⁺-NTA beads (QIAGEN) for histidine tagged proteins or Protein-ASepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed bySDS-PAGE analysis comparing to a known concentration of protein standardby Coomassie blue staining.

The conditioned media from the transfected cells (0.5 to 3 L) isharvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteincomprising the sequence is purified using a Ni²⁺-NTA column (Qiagen).Before purification, imidazole is added to the conditioned media to aconcentration of 5 mM. The conditioned media is pumped onto a 6 mlNi²⁺-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing0.3 M NaCl and 5 mM imidazole at a flow rate of 4–5 ml/min. at 48° C.After loading, the column is washed with additional equilibration bufferand the protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein is then subsequently desaltedinto a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4%mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc containing) constructs of proteins are purified fromthe conditioned media as follows. The conditioned media is pumped onto a5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mMNa phosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 ml of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity of the sequence is assessed by SDS polyacrylamide gels andby N-terminal amino acid sequencing by Edman degradation and otheranalytical procedures as desired or necessary.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

6.11. Example 11 Preparation of Antibodies That Bind PRO

This example illustrates preparation of monoclonal antibodies which canspecifically bind the PRO polypeptide or an epitope on the PROpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO, fusion proteins containing PRO, andcells expressing recombinant PRO on the cell surface. Selection of theimmunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the PRO immunogen emulsified incomplete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1–100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO. Determination of “positive” hybridoma cells secreting the desiredmonoclonal antibodies against PRO is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

6.12. Example 12 Purification of PRO Polypeptides Using SpecificAntibodies

Native or recombinant PRO polypeptides may be purified by a variety ofstandard techniques in the art of protein purification. For example,pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO polypeptide of interest. In general, an immunoaffinity column isconstructed by covalently coupling the anti-PRO polypeptide antibody toan activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PROpolypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PRO polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2–3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

6.13. Example 13 Drug Screening

This invention is particularly useful for screening compounds by usingPRO polypeptides or binding fragment thereof in any of a variety of drugscreening techniques. The PRO polypeptide or fragment employed in such atest may either be free in solution, affixed to a solid support, borneon a cell surface, or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO polypeptide-associated diseaseor disorder. These methods comprise contacting such an agent with an PROpolypeptide or fragment thereof and assaying (I) for the presence of acomplex between the agent and the PRO polypeptide or fragment, or (ii)for the presence of a complex between the PRO polypeptide or fragmentand the cell, by methods well known in the art. In such competitivebinding assays, the PRO polypeptide or fragment is typically labeled.After suitable incubation, free PRO polypeptide or fragment is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular agent to bind to PROpolypeptide or to interfere with the PRO polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO polypeptide, the peptide test compounds arereacted with PRO polypeptide and washed. Bound PRO polypeptide isdetected by methods well known in the art. Purified PRO polypeptide canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. In addition, non-neutralizing antibodies can beused to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

6.14. Example 14 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO polypeptide) orof small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (c.f., Hodgson, Bio/Technology, 9: 19–21 (1991)).

In one approach, the three-dimensional structure of the PRO polypeptide,or of an PRO polypeptide-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry, 31:7796–7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742–746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

6.15. Example 15 Stimulation of Endothelial Cell Proliferation (Assay 8)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to stimulate adrenal corticalcapillary endothelial cell (ACE) growth. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of conditions or disorders where angiogenesiswould be beneficial including, for example, wound healing, and the like(as would agonists of these PRO polypeptides). Antagonists of the PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of cancerous tumors.

Bovine adrenal cortical capillary endothelial (ACE) cells (from primaryculture, maximum of 12–14 passages) were plated in 96-well plates at 500cells/well per 100 microliter. Assay media included low glucose DMEM,10% calf serum, 2 mM glutamine, and 1×penicillin/streptomycin/fungizone.Control wells included the following: (1) no ACE cells added; (2) ACEcells alone; (3) ACE cells plus VEGF (5 ng/ml); and (4) ACE cells plusFGF (5 ng/ml). The control or test sample, (in 100 microliter volumes),was then added to the wells (at dilutions of 1%, 0.1% and 0.01%,respectively). The cell cultures were incubated for 6–7 days at 37°C./5% CO₂. After the incubation, the media in the wells was aspirated,and the cells were washed 1× with PBS. An acid phosphatase reactionmixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1% Triton X-100,10 mM p-nitrophenyl phosphate) was then added to each well. After a 2hour incubation at 37° C., the reaction was stopped by addition of 10microliters 1N NaOH. Optical density (OD) was measured on a microplatereader at 405 nm.

The activity of a PRO polypeptide was calculated as the fold increase inproliferation (as determined by the acid phosphatase activity, OD 405nm) relative to (1) cell only background, and (2) relative to maximumstimulation by VEGF. VEGF (at 3–10 ng/ml) and FGF (at 1–5 ng/ml) wereemployed as-an activity reference for maximum stimulation. Results ofthe assay were considered “positive” if the observed stimulation was≧50% increase over background. VEGF (5 ng/ml) control at 1% dilutiongave 1.24 fold stimulation; FGF (5 ng/ml) control at 1% dilution gave1.46 fold stimulation.

PRO21 tested positive in this assay.

6.16. Example 16 Inhibition of Vascular Endothelial Growth Factor (VEGF)Stimulated Proliferation of Endothelial Cell Growth (Assay 9)

The ability of various PRO polypeptides to inhibit VEGF stimulatedproliferation of endothelial cells was tested. Polypeptides testingpositive in this assay are useful for inhibiting endothelial cell growthin mammals where such an effect would be beneficial, e.g., forinhibiting tumor growth.

Specifically, bovine adrenal cortical capillary endothelial cells (ACE)(from primary culture, maximum of 12–14 passages) were plated in 96-wellplates at 500 cells/well per 100 microliter. Assay media included lowglucose DMEM, 10% calf serum, 2 mM glutamine, and1×penicillin/streptomycin/fungizone. Control wells included thefollowing: (1) no ACE cells added; (2) ACE cells alone; (3) ACE cellsplus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGFplus 5 ng/ml LIF. The test samples, poly-his tagged PRO polypeptides (in100 microliter volumes), were then added to the wells (at dilutions of1%, 0.1% and 0.01%, respectively). The cell cultures were incubated for6–7 days at 37° C./5% CO₂. After the incubation, the media in the wellswas aspirated, and the cells were washed 1× with PBS. An acidphosphatase reaction mixture (100 microliter; 0.1M sodium acetate, pH5.5, 0.1% Triton X-100, 10 mM p-nitrophenyl phosphate) was then added toeach well. After a 2 hour incubation at 37° C., the reaction was stoppedby addition of 10 microliters 1N NaOH. Optical density (OD) was measuredon a microplate reader at 405 nm.

The activity of PRO polypeptides was calculated as the percentinhibition of VEGF (3 ng/ml) stimulated proliferation (as determined bymeasuring acid phosphatase activity at OD 405 nm) relative to the cellswithout stimulation. TGF-beta was employed as an activity reference at 1ng/ml, since TGF-beta blocks 70–90% of VEGF-stimulated ACE cellproliferation. The results are indicative of the utility of the PROpolypeptides in cancer therapy and specifically in inhibiting tumorangiogenesis. Numerical values (relative inhibition) are determined bycalculating the percent inhibition of VEGF stimulated proliferation bythe PRO polypeptides relative to cells without stimulation and thendividing that percentage into the percent inhibition obtained by TGF-βat 1 ng/ml which is known to block 70–90% of VEGF stimulated cellproliferation. The results are considered positive if the PROpolypeptide exhibits 30% or greater inhibition of VEGF stimulation ofendothelial cell growth (relative inhibition 30% or greater).

PRO247, PRO720 and PRO4302 tested positive in this assay.

6.17. Example 17 Enhancement of Heart Neonatal Hypertrophy Induced byLIF+ET-1 (Assay 75)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to enhance neonatal heart hypertrophyinduced by LIF and endothelin-1 (ET-1). A test compound that provides apositive response in the present assay would be useful for thetherapeutic treatment of cardiac insufficiency diseases or disorderscharacterized or associated with an undesired level of hypertrophy ofthe cardiac muscle.

Cardiac myocytes from 1-day old Harlan Sprague Dawley rats (180 μl at7.5×10⁴/ml, serum<0.1, freshly isolated) are introduced on day 1 to96-well plates previously coated with DMEM/F12+4% FCS. Test PROpolypeptide samples or growth medium alone (negative control) are thenadded directly to the wells on day 2 in 20 μl volume. LIF+ET-1 are thenadded to the wells on day 3. The cells are stained after an additional 2days in culture and are then scored visually the next day. A positive inthe assay occurs when the PRO polypeptide treated myocytes obtain ascore greater than zero. A score of zero represents non-responsive cellswhereas scores of 1 or 2 represent enhancement (i.e. they are visuallylarger on the average or more numerous than the untreated myocytes).

PRO21 polypeptides tested positive in this assay.

6.18. Example 18 Detection of Endothelial Cell Apoptosis (FACS) (Assay96)

The ability of PRO polypeptides of the present invention to induceapoptosis in endothelial cells was tested in human venous umbilical veinendothelial cells (HUVEC, Cell Systems) in gelatinized T175 flasks usingHUVEC cells below passage 10. PRO polypeptides testing positive in thisassay are expected to be useful for therapeutically treating conditionswhere apoptosis of endothelial cells would be beneficial including, forexample, the therapeutic treatment of tumors.

On day one, the cells were split [420,000 cells per gelatinized 6 cmdishes—(11×10³cells/cm² Falcon, Primaria)] and grown in media containingserum (CS-C, Cell System) overnight or for 16 hours to 24 hours.

On day 2, the cells were washed 1× with 5 ml PBS; 3 ml of 0% serummedium was added with VEGF (100 ng/ml); and 30 μl of the PRO testcompound (final dilution 1%) or 0% serum medium (negative control) wasadded. The mixtures were incubated for 48 hours before harvesting.

The cells were then harvested for FACS analysis. The medium wasaspirated and the cells washed once with PBS. 5 ml of 1× trypsin wasadded to the cells in a T-175 flask, and the cells were allowed to standuntil they were released from the plate (about 5–10 minutes).Trypsinization was stopped by adding 5 ml of growth media. The cellswere spun at 1000 rpm for 5 minutes at 4° C. The media was aspirated andthe cells were resuspended in 10 ml of 10% serum complemented medium(Cell Systems), 5 μl of Annexin-FITC (BioVison) added and chilled tubeswere submitted for FACS. A positive result was determined to be enhancedapoptosis in the PRO polypeptide treated samples as compared to thenegative control.

PRO4302 polypeptide tested positive in this assay.

6.19. Example 19 Induction of c-fos in HUVEC Cells (Assay 123)

This assay is designed to determine whether PRO polypeptides show theability to induce c-fos in HUVEC cells. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of conditions or disorders where angiogenesiswould be beneficial including, for example, wound healing, and the like(as would agonists of these PRO polypeptides). Antagonists of the PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of cancerous tumors.

Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) ingrowth media (50% Ham's F12 w/o GHT: low glucose, and 50% DMEM withoutglycine: with NaHCO3, 1% glutamine, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF)were plated on 96-well microtiter plates at a cell density of 5×10³cells/well. The day after plating (day 2), the cells were starved for 24hours by removing the growth media and replacing with serum free media.On day 3, the cells are treated with 100 μl/well test samples andcontrols (positive control=growth media; negative control=Protein 32buffer=10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8). One plateof cells was incubated for 30 minutes at 37° C., in 5% CO₂. Anotherplate of cells was incubated for 60 minutes at 37° C., in 5% CO₂. Thesamples were removed, and RNA was harvested using the RNeasy 96 kit(Qiagen). Next, the RNA was assayed for c-fos, egr-1 and GAPDH inductionusing Taqman.

The measure of activity of the fold increase over the negative control(Protein 32/HEPES buffer described above) value was by obtained bycalculating the fold increase of the ratio of c-fos to GAPDH in testsamples as compared to the negative control. The results are consideredpositive if the PRO polypeptide exhibits at least a two-fold value overthe negative buffer control.

PRO1376 polypeptide tested positive in this assay.

6.20. Example 20 Normal Human Iliac Artery Endothelial CellProliferation (Assay 138)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to modulate proliferation of humaniliac artery endothelial cells in culture and, therefore, function asuseful growth or inhibitory factors.

On day 0, human iliac artery endothelial cells (from cell lines, maximumof 12–14 passages) were plated in 96-well plates at 1000 cells/well per100 microliter and incubated overnight in complete media [epithelialcell growth media (EGM, Clonetics), plus supplements: human epithelialgrowth factor (hEGF), bovine brain extract (BBE), hydrocortisone,GA-1000, and fetal bovine serum (FBS, Clonetics)]. On day 1, completemedia was replaced by basal media [EGM plus 1% FBS] and addition of PROpolypeptides at 1%, 0.1% and 0.01%. On day 7, an assessment of cellproliferation was performed by Alamar Blue assay followed by CrystalViolet. Results are expressed as % of the cell growth observed withcontrol buffer.

The following PRO polypeptides stimulated proliferation in this assay:PRO214, PRO256, PRO363, PRO365, PRO791, PRO836, PRO1025, PRO1186,PRO1192, PRO1272, PRO1306, PRO1325, PRO1329, PRO1376, PRO1411, PRO1508,PRO1787, PRO1868, PRO4324, PRO4333, PRO4408, PRO4499, PRO9821, PRO9873,PRO10008, PRO10096, PRO19670, PRO20040, PRO20044 and PRO21384.

The following PRO polypeptides inhibited proliferation in this assay:PRO238, PRO1029, PRO1274, PRO1279, PRO1419, PRO1890, PRO6006 andPRO28631.

6.21. Example 21 Pooled Human Umbilical Vein Endothelial CellProliferation (Assay 139)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to modulate proliferation of pooledhuman umbilical vein endothelial cells in culture and, therefore,function as useful growth or inhibitory factors.

On day 0, pooled human umbilical vein endothelial cells (from celllines, maximum of 12–14 passages) were plated in 96-well plates at 1000cells/well per 100 microliter and incubated overnight in complete media[epithelial cell growth media (EGM, Clonetics), plus supplements: humanepithelial growth factor (hEGF), bovine brain extract (BBE),hydrocortisone, GA-1000, and fetal bovine serum (FBS, Clonetics)]. Onday 1, complete media was replaced by basal media [EGM plus 1% FBS] andaddition of PRO polypeptides at 1%, 0.1% and 0.01%. On day 7, anassessment of cell proliferation was performed by Alamar Blue assayfollowed by Crystal Violet. Results are expresses as % of the cellgrowth observed with control buffer.

The following PRO polypeptides stimulated proliferation in this assay:PRO181, PRO205, PRO221, PRO231, PRO238, PRO241, PRO247, PRO256, PRO258,PRO263, PRO265, PRO295, PRO321, PRO322, PRO337, PRO363, PRO533, PRO697,PRO725, PRO771, PRO788, PRO819, PRO828, PRO846, PRO865, PRO1005,PRO1006, PRO1025, PRO1054, PRO1071, PRO1079, PRO1080, PRO1114, PRO1131,PRO1155, PRO1160, PRO1192, PRO1244, PRO1272, PRO1273, PRO1279, PRO1283,PRO1286, PRO1306, PRO1309, PRO1325, PRO1329, PRO1347, PRO1356, PRO1376,PRO1382, PRO1412, PRO1550, PRO1556, PRO1760, PRO1787, PRO1801, PRO1868,PRO1887, PRO3438, PRO3444, PRO4324, PRO4341, PRO4342, PRO4353, PRO4354,PRO4356, PRO4371, PRO4422, PRO4425, PRO5723, PRO5737, PRO6029, PRO607 1,PRO10096 and PRO21055.

The following PRO polypeptides inhibited proliferation in this assay:PRO229, PRO444, PRO827, PRO1007, PRO1075, PRO1184, PRO1190, PRO1195,PRO1419, PRO1474, PRO1477, PRO1488, PRO1782, PRO4302, PRO4405, PRO5725,PRO5776, PRO7436, PRO9771, PRO10008 and PRO21384.

6.22. Example 22 Human Coronary Artery Smooth Muscle Cell Proliferation(Assay 140)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to modulate proliferation of humancoronary artery smooth muscle cells in culture and, therefore, functionas useful growth or inhibitory factors.

On day 0, human coronary artery smooth muscle cells (from cell lines,maximum of 12–14 passages) were plated in 96-well plates at 1000cells/well per 100 microliter and incubated overnight in complete media[smooth muscle growth media (SmGM, Clonetics), plus supplements:insulin, human epithelial growth factor (hEGF), human fibroblast growthfactor (hFGF), GA-1000, and fetal bovine serum (FBS, Clonetics)]. On day1, complete media was replaced by basal media [SmGM plus 1% FBS] andaddition of PRO polypeptides at 1%, 0.1% and 0.01%. On day 7, anassessment of cell proliferation was performed by Alamar Blue assayfollowed by Crystal Violet. Results are expresses as % of the cellgrowth observed with control buffer.

The following PRO polypeptides stimulated proliferation in this assay:PRO162, PRO182, PRO204, PRO221, PRO230, PRO256, PRO258, PRO533, PRO697,PRO725, PRO738, PRO826, PRO836, PRO840, PRO846, PRO865, PRO982, PRO1025,PRO1029, PRO1071, PRO1083, PRO1134, PRO1160, PRO1182, PRO1184, PRO1186,PRO1192, PRO1274, PRO1279, PRO1283, PRO1306, PRO1308, PRO1325, PRO1337,PRO1338, PRO1343, PRO1376, PRO1387, PRO1411, PRO1412, PRO1415, PRO1434,PRO1474, PRO1550, PRO1556, PRO1567, PRO1600, PRO1754, PRO1758, PRO1760,PRO1787, PRO1865, PRO1868, PRO1917, PRO1928, PRO3438, PRO3562, PRO4333,PRO4345, PRO4353, PRO4354, PRO4408, PRO4430, PRO4503, PRO6714, PRO9771,PRO9820, PRO9940, PRO10096, PRO21055, PRO21184 and PRO21366.

The following PRO polypeptides inhibited proliferation in this assay:PRO181, PRO195, PRO1080, PRO1265, PRO1309, PRO1488, PRO4302, PRO4405 andPRO5725.

6.23. Example 23 Microarray Analysis to Detect Overexpression of PROPolypeptides in HUVEC Cells Treated with Growth Factors

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to induce angiogenesis by stimulatingendothelial cell tube formation in HUVEC cells.

Nucleic acid microarrays, often containing thousands of gene sequences,are useful for identifying differentially expressed genes in tissuesexposed to various stimuli (e.g., growth factors) as compared to theirnormal, unexposed counterparts. Using nucleic acid microarrays, test andcontrol mRNA samples from test and control tissue samples are reversetranscribed and labeled to generate cDNA probes. The cDNA probes arethen hybridized to an array of nucleic acids immobilized on a solidsupport. The array is configured such that the sequence and position ofeach member of the array is known. Hybridization of a labeled probe witha particular array member indicates that the sample from which the probewas derived expresses that gene. If the hybridization signal of a probefrom a test (exposed tissue) sample is greater than hybridization signalof a probe from a control (normal, unexposed tissue) sample, the gene orgenes overexpressed in the exposed tissue are identified. Theimplication of this result is that an overexpressed protein in anexposed tissue may be involved in the functional changes within thetissue following exposure to the stimuli (e.g., tube formation).

The methodology of hybridization of nucleic acids and microarraytechnology is well known in the art. In the present example, thespecific preparation of nucleic acids for hybridization and probes,slides, and hybridization conditions are all detailed in U.S.Provisional Patent Application Ser. No. 60/193,767, filed on Mar. 31,2000 and which is herein incorporated by reference.

In the present example, HUVEC cells grown in either collagen gels orfibrin gels were induced to form tubes by the addition of various growthfactors. Specifically, collagen gels were prepared as describedpreviously in Yang et al., American J. Pathology, 1999, 155(3):887–895and Xin et al., American J. Pathology, 2001, 158(3):1111–1120. Followinggelation of the HUVEC cells, 1× basal medium containing M199supplemented with 1% FBS, 1×ITS, 2 mM L-glutamine, 50 μg/ml ascorbicacid, 26.5 mM NaHCO₃, 100U/ml penicillin and 100 U/ml streptomycin wasadded. Tube formation was elicited by the inclusion in the culture mediaof either a mixture of phorbol myrsitate acetate (50 nM), vascularendothelial cell growth factor (40 ng/ml) and basic fibroblast growthfactor (40 ng/ml) (“PMA growth factor mix”) or hepatocyte growth factor(40 ng/ml) and vascular endothelial cell growth factor (40 ng/ml)(HGF/VEGF mix) for the indicated period of time. Fibrin Gels wereprepared by suspending Huvec (4×10⁵ cells/ml) in M199 containing 1%fetal bovine serum (Hyclone) and human fibrinogen (2.5 mg/ml). Thrombin(50U/ml) was then added to the fibrinogen suspension at a ratio of 1part thrombin solution: 30 parts fibrinogen suspension. The solution wasthen layered onto 10 cm tissue culture plates (total volume: 15ml/plate) and allowed to solidify at 37° C. for 20 min. Tissue culturemedia (10 ml of BM containing PMA (50 nM), bFGF (40ng/ml) and VEGF (40ng/ml)) was then added and the cells incubated at 37° C. in 5% CO₂ inair for the indicated period of time.

Total RNA was extracted from the HUVEC cells incubated for 0, 4, 8, 24,40 and 50 hours in the different matrix and media combinations using aTRIzol extraction followed by a second purification using RNAeasy MiniKit (Qiagen). The total RNA was used to prepare cRNA which was thenhybridized to the microarrays.

In the present experiments, nucleic acid probes derived from the hereindescribed PRO polypeptide-encoding nucleic acid sequences were used inthe creation of the microarray and RNA from the HUVEC cells describedabove were used for the hybridization thereto. Pairwise comparisons weremade using time 0 chips as a baseline. Three replicate samples wereanalyzed for each experimental condition and time. Hence there were 3time 0 samples for each treatment and 3 replicates of each successivetime point. Therefore, a 3 by 3 comparison was performed for each timepoint compared against each time 0 point. This resulted in 9 comparisonsper time point. Only those genes that had increased expression in allthree non-time-0 replicates in each of the different matrix and mediacombinations as compared to any of the three time zero replicates wereconsidered positive. Although this stringent method of data analysisdoes allow for false negatives, it minimizes false positives.

PRO178, PRO195, PRO228, PRO301, PRO302, PRO532, PRO724, PRO730, PRO734,PRO793, PRO871, PRO938, PRO1012, PRO1120, PRO1139, PRO1198, PRO1287,PRO1361, PRO1864, PRO1873, PRO2010, PRO3579, PRO4313, PRO4527, PRO4538,PRO4553, PRO4995, PRO5730, PRO6008, PRO7223, PRO7248 and PRO7261 testedpositive in this assay.

6.24. Example 24 In situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis, and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision, 1: 169–176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A (³³-P)UTP-labeled antisense riboprobe was generatedfrom a PCR product and hybridized at 55° C. overnight. The slides weredipped in Kodak NTB2™ nuclear track emulsion and exposed for 4 weeks.

6.24.1. ³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed-vacuum dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 10 μl each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl RNAsin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. A total of 1.0 μl RQ1DNase was added, followed by incubation at 37° C. for 15 minutes. Atotal of 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) was added, andthe mixture was pipetted onto DE81 paper. The remaining solution wasloaded in a MICROCON-50™ ultrafiltration unit, and spun using program 10(6 minutes). The filtration unit was inverted over a second tube andspun using program 2 (3 minutes). After the final recovery spin, a totalof 100 μl TE was added, then 1 μl of the final product was pipetted onDE81 paper and counted in 6 ml of BIOFLUOR II™.

The probe was run on a TBE/urea gel. A total of 1–3 μl of the probe or 5μl of RNA Mrk III was added to 3 μl of loading buffer. After heating ona 95° C. heat block for three minutes, the gel was immediately placed onice. The wells of gel were flushed, and the sample was loaded and run at180–250 volts for 45 minutes. The gel was wrapped in plastic wrap(SARAN™ brand) and exposed to XAR film with an intensifying screen in a−70° C. freezer one hour to overnight.

6.24.2. ³³P-Hybridization

6.24.2.1. Pretreatment of frozen sections

The slides were removed from the freezer, placed on aluminum trays, andthawed at room temperature for 5 minutes. The trays were placed in a 55°C. incubator for five minutes to reduce condensation. The slides werefixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, andwashed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975ml SQ H₂O). After deproteination in 0.5 μg/ml proteinase K for 10minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmedRNAse-free RNAse buffer), the sections were washed in 0.5×SSC for 10minutes at room temperature. The sections were dehydrated in 70%, 95%,and 100% ethanol, 2 minutes each.

6.24.2.2. Pretreatment of paraffin-embedded sections

The slides were deparaffinized, placed in SQ H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections weredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNase-free RNase buffer; 37° C., 15 minutes) for human embryo tissue, or8× proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30 minutes) forformalin tissues. Subsequent rinsing in 0.5×SSC and dehydration wereperformed as described above.

6.24.2.3. Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4×SSC,50% formamide)—saturated filter paper. The tissue was covered with 50 μlof hybridization buffer (3.75 g dextran sulfate+6 ml SQ H₂O), vortexed,and heated in the microwave for 2 minutes with the cap loosened. Aftercooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC, and 9 ml SQ H₂O wereadded, and the tissue was vortexed well and incubated at 42° C. for 1–4hours.

6.24.2.4. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heatedat 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer was added per slide. After vortexing, 50 μl ³³P mixwas added to 50 μl prehybridization on the slide. The slides wereincubated overnight at 55° C.

6.24.2.5. Washes

Washing was done for 2×10 minutes with 2×SSC, EDTA at room temperature(400 ml 20×SSC+16 ml 0.25 M EDTA, V_(f)=4L), followed by RNAseAtreatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnasebuffer=20 μg/ml), The slides were washed 2×10 minutes with 2×SSC, EDTAat room temperature. The stringency wash conditions were as follows: 2hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V_(f)=4L).

6.24.2.6. Oligonucleotides

In situ analysis was performed on three of the DNA sequences disclosedherein. The primers used to generate the probes and/or the probesemployed for these analyses are as follows:

DNA33100-p1: 5′GGA TTC TAA TAC GAC TCA CTA TAG GGC CGG GTG GAG GTG GAACAG AAA3′ (SEQ ID NO:375) DNA33100-p2: 5′CTA TGA AAT TAA CCC TCA CTA AAGGGA CAC AGA CAG AGC CCC ATA CGC3′ (SEQ ID NO:376) DNA34431-p1: 5′GGA TTCTAA TAC GAC TCA CTA TAG GGC CAG GGA AAT CCG GAT GTC TC 3′ (SEQ IDNO:377) DNA34431-p2: 5′CTA TGA AAT TAA CCC TCA CTA AAG GGA GTA AGG GGATGC CAC CGA GTA3′ (SEQ ID NO:378) DNA38268-p1: 5′GGA TTC TAA TAC GAC TCACTA TAG GGC CAG CTA CCC GCA GGA GGA GG 3′ (SEQ ID NO:379) DNA38268-p2:5′CTA TGA AAT TAA CCC TCA CTA AAG GGA TCC CAG GTG ATG AGG TCC AGA3′ (SEQID NO:380) DNA64908 probe:5′CCATCTCGGAGACCTTTGTGCAGCGTGTATACCAGCCTTACCTCACCACTTGCGACGGACACAGAG(SEQ ID NO:381)CCTGCAGCACCTACCGAACCATCTACCGGACTGCCTATCGCCGTAGCCCTGGGGTGACTCCCGCAAGCCTCGCTATGCTTGCTGCCCTGGTTGGAAGAGGACCAGTGGGCTCCCTGGGGCTTGTGGAGCAGCAATATGCCAGCCTCCATGTGGGAATGGAGGGAGTTGCATCCGCCCAGGACACTGCCGCTGCCCTGTGGGATGGCAGGGAGATACTTGCCAGACAGATGTTGATGAATGCAGTACAGGAGAGGCCAGTTGTCCCCAGCGCTGTGTCAATACTGTGGGAAGTTACTGGTGCCAGGGATGGGAGGGACAAAGCCCATCTGCAGATGGGACGCGCTGCCTGTCTAAGGAGGGGCCCTCCCGGTGGCCCCAACCCCACAGCAGGAGTGGACAGCA3′

6.24.2.7. Results

In situ analysis was performed and the results from these analyses areas follows:

6.24.2.7.1. DNA33100-1159 (PRO229) (Scavenger-R/CD6 homolog TNF motif)

A specific positive signal was observed in mononuclear phagocytes(macrophages) of fetal and adult spleen, liver, lymph node and thymus.All other tissues were negative.

6.24.2.7.2. DNA34431-1177 (PRO263) (CD44)

A specific positive signal was observed in human fetal tissues andplacenta over mononuclear cells, with strong expression in epithelialcells of the adrenal cortex. All adult tissues were negative.

6.24.2.7.3. DNA38268-1188 (PRO295) (Integrin)

A specific positive signal was observed in human fetal ganglion cells,fetal neurons, adult adrenal medulla and adult neurons. All othertissues were negative.

6.24.2.7.4. DNA64908-1163-1 (PRO1449)

A specific positive signal was observed in the developing vasculature(from E7-E11), in endothelial cells and in progenitors of endothelialcells in wholemount in situ hybridizations of mouse embryos (FIG. 375).Specific expression was also observed in a subset of blood vessels andepidermis from E12 onward. A mouse orthologue of PRO1449 which has about78% amino acid identity with PRO1449 was used as the probe.

In normal adult tissues, expression was low to absent. When present,expression was confined to the vasculature (FIG. 376). FIG. 376 furthershows that highest expression in adult tissues was observed regionallyin vessels running within the white matter of the brain. Elevatedexpression was also observed in vasculature of many inflamed anddiseased tissues, including, but not limited to, tumor vasculature.

Following electroporation of the mouse orthologue of PRO1449 into thechoroid layer in the eyes of chicken embryos, new vessel formation wasobserved in the electroporated eye (top right), but not in the controlside from the same embryo (top left), or an embryo that waselectroporated with a control cDNA (bottom right) (FIG. 377).

6.25. Example 25 Inhibition of Basic Fibroblast Growth Factor (bFGF)Stimulated Proliferation of Endothelial Cell Growth

The ability of various PRO polypeptides to inhibit bFGF stimulatedproliferation of endothelial cells was tested. Polypeptides testingpositive in this assay are useful for inhibiting endothelial cell growthin mammals where such an effect would be beneficial, e.g., forinhibiting tumor growth.

Specifically, human venous umbilical vein endothelial cells (HUVEC, CellSystems) in epithelial cell growth media (EGM, Clonetics) were plated on96-well microtiter plates at a cell density of 5×10³ cells/well in avolume of 100 μl/well. The day after plating (day 2), the cells werestarved for 24 hours by removing the growth media and replacing withstarving media (M199 supplemented with 1% FBS, 2 mM L-glutamine, 100U/mlpenicillin and 100 U/ml streptomycin). On day 5, the cells are treatedwith either: (1) M199 with 10% FBS; (2) M199 with 1% FBS; (3) M199 with1% FBS and 20 ng/ml bFGF; (4) M199 with 1% FBS and 20 ng/ml bFGF and PROpolypeptide (500 nM); (5) M199 with 1% FBS and 20 ng/ml bFGF and PROpolypeptide (50 nM); or (6) M199 with 1% FBS and 20 ng/ml bFGF and PROpolypeptide (5 nM). On day 8, an assessment of cell proliferation wasperformed by Alamar Blue assay. Optical density (OD) was measured on amicroplate reader at excitation 530 and emission at 590 nm.

The activity of PRO polypeptides was calculated as the percentinhibition of bFGF stimulated proliferation relative to the cellswithout stimulation. The results are indicative of the utility of thePRO polypeptides in cancer therapy and specifically in inhibiting tumorangiogenesis. Numerical values (relative inhibition) are determined bycalculating the percent inhibition of bFGF stimulated proliferation bythe PRO polypeptides relative to cells without stimulation. The resultsare considered positive if the PRO polypeptide exhibits 30% or greaterinhibition of bFEGF stimulation of endothelial cell growth.

PRO5725 tested positive in this assay.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct(s) deposited,since the deposited embodiment(s) is/are intended as singleillustration(s) of certain aspects of the invention and any constructsthat are functionally equivalent are within the scope of this invention.The deposit of material(s) herein does not constitute an admission thatthe written description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated antibody that specifically binds to the polypeptide ofSEQ ID NO:346.
 2. The antibody of claim 1 which is a monoclonalantibody.
 3. The antibody of claim 1 which is a humanized antibody. 4.The antibody of claim 1 which is an antibody or a fragment thereof. 5.The antibody of claim 1 which is labeled.